Compositions comprising a bacterial strain of the genus megasphera and uses thereof

ABSTRACT

The invention provides compositions comprising bacterial strains for treating and preventing neurodegenerative disorders.

CROSS-REFERENCE

This application is a continuation of International Application No.PCT/EP2018/065858, filed Jun. 14, 2018, which claims the benefit ofGreat Britain Application No. 1709468.1, filed Jun. 14, 2017, GreatBritain Application No. 1709534.0, filed Jun. 15, 2017, Great BritainApplication No. 1712851.3, filed Aug. 10, 2017, Great BritainApplication No. 1803826.5, filed Mar. 9, 2018, Great Britain ApplicationNo. 1805989.9, filed Apr. 11, 2018, Great Britain Application No.1805990.7, filed Apr. 11, 2018, Great Britain Application No. 1805991.5,filed Apr. 11, 2018, Great Britain Application No. 1806779.3, filed Apr.25, 2018, Great Britain Application No. 1806780.1, filed Apr. 25, 2018,all of which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ANSI format and is hereby incorporated byreference in its entirety. Said ANSI copy, created on Jan. 21, 2020, isnamed 56708-721_301_SL.txt and is 14,775 bytes in size.

TECHNICAL FIELD

This invention is in the field of compositions comprising bacterialstrains isolated from the mammalian digestive tract and the use of suchcompositions in the treatment of disease.

BACKGROUND TO THE INVENTION

The human intestine is thought to be sterile in utero, but it is exposedto a large variety of maternal and environmental microbes immediatelyafter birth. Thereafter, a dynamic period of microbial colonization andsuccession occurs, which is influenced by factors such as delivery mode,environment, diet and host genotype, all of which impact upon thecomposition of the gut microbiota, particularly during early life.Subsequently, the microbiota stabilizes and becomes adult-like [1]. Thehuman gut microbiota contains more than 500-1000 different phylotypesbelonging essentially to two major bacterial divisions, theBacteroidetes and the Firmicutes [2]. The successful symbioticrelationships arising from bacterial colonization of the human gut haveyielded a wide variety of metabolic, structural, protective and otherbeneficial functions. The enhanced metabolic activities of the colonizedgut ensure that otherwise indigestible dietary components are degradedwith release of by-products providing an important nutrient source forthe host. Similarly, the immunological importance of the gut microbiotais well-recognized and is exemplified in germfree animals which have animpaired immune system that is functionally reconstituted following theintroduction of commensal bacteria [3-5].

Dramatic changes in microbiota composition have been documented ingastrointestinal disorders such as inflammatory bowel disease (IBD). Forexample, the levels of Clostridium cluster XIVa bacteria are reduced inIBD patients whilst numbers of E. coli are increased, suggesting a shiftin the balance of symbionts and pathobionts within the gut [6-9].

In recognition of the potential positive effect that certain bacterialstrains may have on the animal gut, various strains have been proposedfor use in the treatment of various diseases (see, for example,[10-13]). Also, certain strains, including mostly Lactobacillus andBifidobacterium strains, have been proposed for use in treating variousinflammatory and autoimmune diseases that are not directly linked to theintestines (see [14] and [15] for reviews). However, the relationshipbetween different diseases and different bacterial strains, and theprecise effects of particular bacterial strains on the gut and at asystemic level and on any particular types of diseases are poorlycharacterised, particularly for neurodegenerative disorders.

Recently, there has been increased interest in the art regardingalterations in the gut microbiome that may play a pathophysiologicalrole in human brain diseases [16]. Preclinical and clinical evidence arestrongly suggesting a link between brain development and microbiota[17]. A growing body of preclinical literature has demonstratedbidirectional signalling between the brain and the gut microbiome,involving multiple neurocrine and endocrine signalling systems. Indeed,increased levels of Clostridium species in the microbiome have beenlinked to brain disorders [18], and an imbalance of the Bacteroidetesand Finnicutes phyla has also been implicated in brain developmentdisorders [19]. Suggestions that altered levels of gut commensals,including those of Bifidobacterium, Lactobacillus, Sutterella,Prevotella and Ruminococcus genera and of the Alcaligenaceae family areinvolved in immune-mediated central nervous system (CNS) disorders, arequestioned by studies suggesting a lack of alteration in the microbiotabetween patients and healthy subjects [19]. There have also beensuggestions that the administration of probiotics may be beneficial inthe treatment of neurological disorders. However, these studies failedto conclude that probiotic compositions per se can achieve therapeuticbenefits with respect to the treatment of neurodegeneration and did notshow any useful effects for any particular bacteria [20, 21]. Thisindicates that, at present, the practical effect of the link between themicrobiome and human brain diseases is poorly characterised.Accordingly, more direct analytical studies are required to identify thetherapeutic impact of altering the microbiome on neurodegenerativedisorders.

There is a requirement in the art for new methods of treatingneurodegenerative disorders. There is also a requirement for thepotential effects of gut bacteria to be characterised so that newtherapies using gut bacteria can be developed.

SUMMARY OF THE INVENTION

The inventors have developed new therapies for treating and preventingneurodegenerative disorders. The inventors have identified thatbacterial strains from the genus Megasphaera may be effective fortreating neurodegenerative diseases. As described in the examples,administration of compositions comprising Megasphaera massiliensis canprotect against reactive oxygen species and prevent inflammation, thusacting as a neuroprotectant. The inventors have also identified thattreatment with Megasphaera massiliensis can reduce the activation ofproinflammatory molecules, such as NFκB and IL-6, by LPS and mutantα-synuclein. The inventors have identified that treatment withMegasphaera massiliensis can reduce histone deacetylation activity andlipid peroxidation in vitro, which can help to reduce cell death andapoptosis. The inventors have also identified that Megasphaeramassiliensis can produce indole that can attenuate inflammation andoxidative stress. Furthermore, the inventors have demonstrated thattreatment with Megasphaera massiliensis can increase kynurenine levels.

The inventors have also identified that Megasphaera massiliensisproduces certain organic acids including hexanoic acid, valeric acid and4-hydroxyphenylacetic acid. The inventors have also found thatMegasphaera massiliensis can increase the activation of thepro-inflammatory cytokine IL-8, which can help to promote neuronmyelination. The inventors have also identified that treatment with acombination of Megasphaera massiliensis and retinoic acid can increasethe secretion of brain-derived neurotrophic factor (BDNF), which canhelp promote neurogenesis and neuritogenesis and/or prevent cell death.The inventors have also identified that treatment with Megasphaeramassiliensis, which can produce valeric acid, can reduce histonedeacetylation, which can help to reduce cell death and apoptosis.Furthermore, the inventors have also found that Megasphaera massiliensiscan produce hexanoic acid, which can be neuroprotective orneurorestorative, for example by promoting neurite outgrowth. Theinventors have found that Megasphaera massiliensis that can producehexanoic acid increase the expression of MAP2 (Microtubule-associatedprotein 2), which is thought to be essential for microtubule formationin neuritogenesis. Therefore, the inventors have found that Megasphaeramassiliensis that can produce hexanoic acid can be used to promoteneurite outgrowth. Megasphaera massiliensis and other bacteria thatproduce organic acids like hexanoic acid, valeric acid and4-hydroxyphenylacetic acid may therefore be useful for treatingneurodegenerative disorders.

In a first embodiment, the invention provides a composition comprising abacterial strain of the genus Megasphaera, for use in therapy, such asfor use in a method of treating or preventing a neurodegenerativedisorder.

In particular embodiments, the invention provides a compositioncomprising a bacterial strain of the genus Megasphaera, for use in amethod of treating or preventing a disease or condition selected fromthe group consisting of: Parkinson's disease, including progressivesupranuclear palsy, progressive supranuclear palsy,Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus,vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism;Alzheimer's disease, including Benson's syndrome; multiple sclerosis;Huntington's disease; amyotrophic lateral sclerosis; Lou Gehrig'sdisease; motor neurone disease; prion disease; spinocerebellar ataxia;spinal muscular atrophy; dementia, including Lewy body, vascular andfrontotemporal dementia; primary progressive aphasia; mild cognitiveimpairment; HIV-related cognitive impairment and corticobasaldegeneration.

In preferred embodiments, the invention provides a compositioncomprising a bacterial strain of the genus Megasphaera, for use in amethod of treating or preventing Parkinson's disease, such asenvironmental, familial or Parkinson's associated with generalinflammatory status. The inventors have identified that treatment withMegasphaera strains can reduce the activation of proinflammatorymolecules, such as NFκB and IL-6, by LPS and mutant α-synuclein in invitro models of environmental and familial Parkinson's. In preferredembodiments, the invention provides a composition comprising a bacterialstrain of the species Megasphaera massiliensis, for use in the treatmentof Parkinson's disease. Compositions using Megasphaera massiliensis maybe particularly effective for treating Parkinson's.

In some embodiments, the compositions of the invention are for use in amethod of treating or preventing early-onset neurodegenerative disease.In some embodiments, the compositions of the invention are for use in amethod of preventing or delaying onset or progression of aneurodegenerative disorder.

In preferred embodiments of the invention, the bacterial strain in thecomposition is of Megasphaera massiliensis. Closely related strains mayalso be used, such as bacterial strains that have a 16S rRNA sequencethat is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical tothe 16S rRNA sequence of a bacterial strain of Megasphaera massiliensis.Preferably, the bacterial strain has a 16S rRNA sequence that is atleast 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:1or 2. Preferably, the sequence identity is to SEQ ID NO:2. Preferably,the bacterial strain for use in the invention has the 16S rRNA sequencerepresented by SEQ ID NO:2.

In certain embodiments, the composition of the invention is for oraladministration. Oral administration of the strains of the invention canbe effective for neurodegenerative disorders. Also, oral administrationis convenient for patients and practitioners and allows delivery toand/or partial or total colonisation of the intestine.

In certain embodiments, the composition of the invention comprises oneor more pharmaceutically acceptable excipients or carriers.

In certain embodiments, the composition of the invention comprises abacterial strain that has been lyophilised. Lyophilisation is aneffective and convenient technique for preparing stable compositionsthat allow delivery of bacteria.

In certain embodiments, the invention provides a food product comprisingthe composition as described above.

In certain embodiments, the invention provides a vaccine compositioncomprising the composition as described above.

Additionally, the invention provides a method of treating or preventingneurodegenerative disorders, comprising administering a compositioncomprising a bacterial strain of the genus Megasphaera.

In developing the above invention, the inventors have identified andcharacterised a bacterial strain that is particularly useful fortherapy. The Megasphaera massiliensis strain of the invention is shownto be effective for treating the diseases described herein, such asneurodegenerative diseases. Therefore, in another aspect, the inventionprovides a cell of the Megasphaera massiliensis strain deposited underaccession number NCIMB 42787, or a derivative thereof. The inventionalso provides compositions comprising such cells, or biologically purecultures of such cells. The invention also provides a cell of theMegasphaera massiliensis strain deposited under accession number NCIMB42787, or a derivative thereof, for use in therapy, in particular forthe diseases described herein.

In certain embodiments of the invention, the composition is for use intreating brain injury. The neuroprotective activity of the compositionsof the invention and their ability to reduce levels of histonedeacetylase activity (HDAC) may make them useful for treating braininjury. In preferred embodiments, the compositions of the invention arefor use in treating stroke, such as treating brain injury resulting froma stroke.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Cell viability of neuroblastoma cells

FIG. 2: Down-regulation of IL-6 secretion

FIG. 3 Secretion of IL-8

FIG. 4A: Inhibition of α-synuclein IL-6; and FIG. 4B: Inhibition ofα-synuclein IL-8 secretion

FIG. 5: Inhibition of α-synuclein induced NFκB promoter activation

FIG. 6: Inhibition of LPS induced NFκB promoter activation

FIG. 7: Change in antioxidant capacity

FIG. 8: Change in total anti-oxidant capacity (lipid oxidation)

FIG. 9: Change in histone deacetylase (HDAC) activity

FIG. 10: Level of Indole production

FIG. 11: Level of Kyrunenine production

FIGS. 12A-12C: Mean Dopamine (DA) levels (FIG. 12A), DOPAC levels (FIG.12B) and HVA levels (FIG. 12C) in striatum. Data is displayed asMean+SEM.

FIGS. 13A-13B: Promoting neurite outgrowth: light microscopy and MAP2gene expression (FIG. 13A), Phalloidin immunofluorescence microscopy(FIG. 13B)

FIGS. 14A-14B: Change in ROS levels in (FIG. 14A) U373 cells and (FIG.14B) SHSY-5Y cells

FIG. 15: Neuroprotection—cell viability. FIG. 15 shows the same data asFIG. 1.

FIGS. 16A-16C: Strain-induced changes in whole cell and cell lysatehistone deacetylase activity (FIG. 16A), acid-induced changes in histonedeacetylase activity (FIG. 16B), metabolite production by strains (FIG.16C)

FIGS. 17A-17C: HDAC1 inhibition (FIG. 17A), HDAC2 inhibition (FIG. 17B),HDAC3 inhibition (FIG. 17C)

FIGS. 18A-18C: Inhibition of Class I HDACs (FIG. 18A); inhibition ofHDAC1 (FIG. 18B); inhibition of HDAC2 (FIG. 18C); inhibition of HDAC3(FIG. 18D)

FIG. 19: Level of BDNF production

FIG. 20: Levels of metabolite production—neurotransmitters in the brain

FIG. 21: Levels of metabolite production—organic acids in thesupernatant

FIGS. 22A-22F: Effect on intestinal barrier function. IL-8 (FIG. 22A),TJ1, TJP2, Occludin and Villin (FIG. 22B), mRNA expression of TJP 1,Colon and Ileum (FIG. 22C), mRNA expression of Occludin, Colon and Ileum(FIG. 22D), Permeability in the Ileum (FIG. 22E), Permeability in thecolon (FIG. 22F)

FIG. 23: Production of neurotransmitters in the brain

FIGS. 24A-24D: Changes in Hippocampal Receptor Expression—FIG. 24A)Oxytocin Receptor, FIG. 24B) Vasopressin Receptor, FIG. 24C)Glucocorticoid Receptor and FIG. 24D) Mineralocorticoid Receptor

FIGS. 25A-25C: Changes in Hippocampal Expression of FIG. 25A)Corticotropin-Releasing Hormone (CRH), FIG. 25B) BDNF Expression andFIG. 25C) TLR4

FIGS. 26A-26B: FIG. 26A) Changes in Hippocampal Corticotropin ReleasingHormone Receptor 1 (CRFR1) Expression and FIG. 26B) CorticotropinReleasing Hormone Receptor 2 (CRFR2) Expression

FIGS. 27A-27C: Changes in Hippocampal Expression of FIG. 27A) TumourNecrosis Factor, FIG. 27B) Interleukin 1b and FIG. 27C) IL-6

FIGS. 28A-28B: FIG. 28A) Changes in Hippocampal Integrin Alpha M (CD11b)Expression and FIG. 28B) Changes in Hippocampal Serotonin 1A Receptor(5-HT1A receptor) Expression

FIGS. 29A-29B: FIG. 29A) Changes in Hippocampal Glutamate IonotropicReceptor NMDA Type Subunit 2A (Grin2A) and FIG. 29B) GlutamateIonotropic Receptor NMDA Type Subunit 2B (Grin2B) expression

FIGS. 30A-30C: Changes in Hippocampal Expression of FIG. 30A)Gamma-Aminobutyric Acid A Receptor 2 (GABA A2), FIG. 30B)Gamma-Aminobutyric Acid B Receptor 1 (GABA BR1) and

FIG. 30C) Dopamine Receptor 1 (DRD1)

FIGS. 31A-31D: Changes in Amygdala Receptor Expression—FIG. 31A)Oxytocin Receptor, FIG. 31B) Vasopressin Receptor, FIG. 31C)Glucocorticoid Receptor and FIG. 31D) Mineralocorticoid Receptor

FIGS. 32A-32D: Changes in Amygdala Expression of FIG. 32A) Brain DerivedNeurotrophic Factor (BDNF), FIG. 32B) Toll-like Receptor 4 (TLR-4), FIG.32C) Corticotropin Releasing Hormone Receptor 1 (CRFR1) and FIG. 32D)Corticotropin Releasing Hormone Receptor 2 (CRFR2)

FIGS. 33A-33D: Changes in Amygdala Expression of FIG. 33A) IntegrinAlpha M (CD11b), FIG. 33B) Interleukin-6 (IL-6), FIG. 33C) GlutamateIonotropic Receptor NMDA Type Subunit 2A (Grin2A) and FIG. 33D)Glutamate Ionotropic Receptor NMDA Type Subunit 2B (Grin2B)

FIGS. 34A-34C: Changes in Amygdala Expression of FIG. 34A) GABA-AReceptor Alpha 2 Subunit (GABRA2), FIG. 34B) GABA-A Type B Receptor 1Subunit (GABBR1) and FIG. 34C) Dopamine Receptor 1 (DRD1)

FIGS. 35A-35D: Changes in Prefrontal Cortex Expression of FIG. 35A)Oxytocin Receptor, FIG. 35B) Brain Derived Neurotrophic Factor (BDNF),FIG. 35C) Mineralocorticoid Receptor and FIG. 35D) GlucocorticoidReceptor

FIGS. 36A-36D: Changes in Prefrontal Cortex Expression of FIG. 36A)Toll-like Receptor 4 (TLR-4), FIG. 36B) Corticotropin Releasing HormoneReceptor 1 (CRFR1), FIG. 36C) Corticotropin Releasing Hormone Receptor 2(CRFR2) and FIG. 36D) Integrin Alpha M (CD11b)

FIGS. 37A-37D: Changes in Prefrontal Cortex Expression of FIG. 37A)Interleukin-6 (IL-6), FIG. 37B) Glutamate Ionotropic Receptor NMDA TypeSubunit 2A (Grin2A), FIG. 37C) Glutamate Ionotropic Receptor NMDA TypeSubunit 2B (Grin2B) and FIG. 37D) GABA-A Receptor Alpha 2 Subunit(GABRA2)

FIGS. 38A-38B: Changes in Prefrontal Cortex Expression of FIG. 38A)GABA-A Receptor Type B Receptor Subunit 1 (GABBR1) and FIG. 38B)Dopamine Receptor 1 (DRD1)

FIGS. 39A-39B: Changes in Colon Expression of FIG. 39A) TryptophanHydroxylase-1 (Tph1) and FIG. 39B) Indoleamine2,3-Dioxygenase-1 (IDO1)

FIGS. 40A-40B: Changes in Ileum Expression of FIG. 40A) TryptophanHydroxylase-1 (Tph1) and FIG. 40B) Indoleamine2,3-Dioxygenase-1 (IDO1)

FIGS. 41A-41C: Changes in Circulating Tryptophan Metabolite Levels FIG.41A) Kynurenine, FIG. 41B) Tryptophan and FIG. 41C)Kynurenine/Tryptophan Index of metabolism

FIG. 42: Effect on Interferon-γ Production from mouse Splenocytes frommice fed with MRx0029

FIG. 43: Effect on Interleukin-1β Production from Splenocytes

FIG. 44: Effect on Interleukin-6 Production from Splenocytes

FIG. 45: Effect on Tumour Necrosis Factor Production from Splenocytes

FIG. 46: Effect on Interleukin-10 Production from Splenocytes

FIG. 47: Effect on Chemoattractant CXCL1 Production from Splenocytes

FIG. 48: Changes in Caecal Short Chain Fatty Acid Levels

FIG. 49: MRx0029 and MRX005-induced changes in gene expression levels ofActin, Villin, Occludin TJP1, TJP2, MAP2, DRD2, GABRB3, SYP, PINK1,PARK7 and NSE.

FIG. 50: SHSY5Y cell differentiation induced by MRx0005 and MRx0029.(A-C) Representative images of immuno labelled cells with Phalloidin andMAP2. (D-F) images of A-C merged with DAPI images. (G-I) (33 tubulinimmunolabelled cells. (J-L) merged with DAPI images. Magnification ×630.Western blot analysis of effects of MRx0005 and MRx0029 treatment onSHSY5Y cells. Western blot membranes were probed with antibodies to MAP2(M) and b3 tubulin (N). Actin was used as a loading control. Lowerpanels: representative blots from one of six separate experiments; upperpanels: relative densitometric intensity.

DISCLOSURE OF THE INVENTION Bacterial Strains

The compositions of the invention comprise a bacterial strain of thegenus Megasphaera. The examples demonstrate that bacteria of this genusare useful for treating or preventing neurodegenerative disorders. Thepreferred bacterial strains are of the species Megasphaera massiliensis.

Examples of Megasphaera species for use in the invention includeMegasphaera elsdenii, Megasphaera cerevisiae, Megasphaera massiliensis,Megasphaera indica, Megasphaera paucivorans, Megasphaera sueciensis andMegasphaera micronuciformis. A further example of a Megasphaera speciesfor use in the invention is Megasphaera hexanoica. The Megasphaera areobligately anaerobic, lactate-fermenting, gastrointestinal microbe ofruminant and non-ruminant mammals, including humans.

The type strain of M massiliensis is NP3 (=CSUR P245=DSM 26228)[22]. TheGenBank accession number for the 16S rRNA gene sequences of Mmassiliensis strain NP3 is JX424772.1 (disclosed herein as SEQ ID NO:1).

The Megasphaera massiliensis bacterium tested in the Examples isreferred to herein as strain MRx0029. A 16S rRNA sequence for theMRx0029 strain that was tested is provided in SEQ ID NO:2.

Strain MRx0029 was deposited with the international depositary authorityNCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) by 4DPharma Research Ltd. (Life Sciences Innovation Building, Cornhill Road,Aberdeen, AB25 2ZS, Scotland) on 13 Jul. 2017 as “Megasphaeramassiliensis MRx0029” and was assigned accession number NCIMB 42787.

Bacterial strains closely related to the strain tested in the examplesare also expected to be effective for treating or preventingneurodegenerative disorders. In certain embodiments, the bacterialstrain for use in the invention has a 16S rRNA sequence that is at least95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16S rRNAsequence of a bacterial strain of Megasphaera massiliensis. Preferably,the bacterial strain for use in the invention has a 16S rRNA sequencethat is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical toSEQ ID NO:1 or 2. Preferably, the sequence identity is to SEQ ID NO:2.Preferably, the bacterial strain for use in the invention has the 16SrRNA sequence represented by SEQ ID NO:2.

Bacterial strains that are biotypes of strains MRx0029 or NP3 are alsoexpected to be effective for treating or preventing neurodegenerativedisorders. A biotype is a closely related strain that has the same orvery similar physiological and biochemical characteristics.

Strains that are biotypes of strains MRx0029 or NP3 and that aresuitable for use in the invention may be identified by sequencing othernucleotide sequences for strains MRx0029 or NP3. For example,substantially the whole genome may be sequenced and a biotype strain foruse in the invention may have at least 95%, 96%, 97%, 98%, 99%, 99.5% or99.9% sequence identity across at least 80% of its whole genome (e.g.across at least 85%, 90%, 95% or 99%, or across its whole genome). Othersuitable sequences for use in identifying biotype strains may includehsp60 or repetitive sequences such as BOX, ERIC, (GTG)₅ (SEQ ID NO: 38),or REP or [23]. Biotype strains may have sequences with at least 95%,96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to thecorresponding sequence of the strains MRx0029 or NP3.

Alternatively, strains that are biotypes of strains MRx0029 or NP3 andthat are suitable for use in the invention may be identified by usingstrains MRx0029 or NP3 and restriction fragment analysis and/or PCRanalysis, for example by using fluorescent amplified fragment lengthpolymorphism (FAFLP) and repetitive DNA element (rep)-PCRfingerprinting, or protein profiling, or partial 16S or 23S rDNAsequencing. In preferred embodiments, such techniques may be used toidentify other Megasphaera massiliensis strains.

In certain embodiments, strains that are biotypes of strains MRx0029 orNP3 and that are suitable for use in the invention are strains thatprovide the same pattern as strains MRx0029 or NP3 when analysed byamplified ribosomal DNA restriction analysis (ARDRA), for example whenusing Sau3AI restriction enzyme (for exemplary methods and guidance see,for example, [24]). Alternatively, biotype strains are identified asstrains that have the same carbohydrate fermentation patterns as strainsMRx0029 or NP3.

Other Megasphaera strains that are useful in the compositions andmethods of the invention, such as biotypes of strains MRx0029 or NP3,may be identified using any appropriate method or strategy, includingthe assays described in the examples. For instance, strains for use inthe invention may be identified by culturing with neuroblastoma cellsand then assessing cytokine levels and levels of neuroprotection orneuroproliferation. In particular, bacterial strains that have similargrowth patterns, metabolic type and/or surface antigens to strainsMRx0029 or NP3 may be useful in the invention. A useful strain will havecomparable immune modulatory activity to strains MRx0029 or NP3. Inparticular, a biotype strain will elicit comparable effects on theneurodegenerative disease models and comparable effects on cytokinelevels to the effects shown in the Examples, which may be identified byusing the culturing and administration protocols described in theExamples.

A particularly preferred strain of the invention is the Megasphaeramassiliensis MRx0029 strain. This is the exemplary strain tested in theexamples and shown to be effective for treating disease. Therefore, theinvention provides a cell, such as an isolated cell, of the Megasphaeramassiliensis strain MRx0029, or a derivative thereof. The invention alsoprovides a composition comprising a cell of the Megasphaera massiliensisstrain MRx0029, or a derivative thereof. The invention also provides abiologically pure culture of the Megasphaera massiliensis strainMRx0029. The invention also provides a cell of the Megasphaeramassiliensis strain MRx0029, or a derivative thereof, for use intherapy, in particular for the diseases described herein.

A particularly preferred strain of the invention is the Megasphaeramassiliensis strain deposited under accession number NCIMB 42787. Thisis the exemplary MRx0029 strain tested in the examples and shown to beeffective for treating disease. Therefore, the invention provides acell, such as an isolated cell, of the Megasphaera massiliensis straindeposited under accession number NCIMB 42787, or a derivative thereof.The invention also provides a composition comprising a cell of theMegasphaera massiliensis strain deposited under accession number NCIMB42787, or a derivative thereof. The invention also provides abiologically pure culture of the Megasphaera massiliensis straindeposited under accession number NCIMB 42787. The invention alsoprovides a cell of the Megasphaera massiliensis strain deposited underaccession number NCIMB 42787, or a derivative thereof, for use intherapy, in particular for the diseases described herein.

A derivative of the strain of the invention may be a daughter strain(progeny) or a strain cultured (subcloned) from the original. Aderivative of a strain of the invention may be modified, for example atthe genetic level, without ablating the biological activity. Inparticular, a derivative strain of the invention is therapeuticallyactive. A derivative strain will have comparable therapeutic activity tothe MRx0029 strain. In particular, a derivative strain will elicitcomparable effects on the neurodegenerative disease models andcomparable effects on cytokine levels to the effects shown in theExamples, which may be identified by using the culturing andadministration protocols described in the Examples. A derivative of theMRx0029 strain will generally be a biotype of the MRx0029 strain.

References to cells of the Megasphaera massiliensis MRx0029 strainencompass any cells that have the same safety and therapeutic efficacycharacteristics as the strain MRx0029, and such cells are encompassed bythe invention.

In preferred embodiments, the bacterial strains in the compositions ofthe invention are viable and capable of partially or totally colonisingthe intestine.

The inventors have found that Megasphaera massiliensis strains reducethe activation of inflammatory cytokines such as IL-6 and increase theactivation of the inflammatory cytokine IL-8. IL-8 has been implicatedin myelin sheath formation [25]. Chronic inflammation induced by IL-6can ultimately lead to cell death. Therefore, the bacterial strains ofthe invention are particularly useful in the treatment or prevention ofneurodegenerative disorders. In some embodiments, the bacterial strainsare useful in the treatment of conditions characterised by the enhancedactivation of IL-6. In some embodiments, the compositions of theinvention are for use in the treatment or prevention ofneurodegenerative diseases characterised by demyelination. Manyneurodegenerative diseases are characterised by demyelination.Demyelination impedes the propagation of action potentials withinneurons, impairing effective communication within the nervous system.IL-8 has been shown to contribute positively to myelin sheath formationand repair. Therefore, the compositions of the invention areparticularly beneficial in the treatment or prevention ofneurodegenerative disorders characterised by demyelination, such asMultiple Sclerosis.

The inventors have found that the Megasphaera massiliensis strains ofthe invention alleviate symptoms of neurodegenerative diseases in modelsof the disease. For example, the inventors have found that theMegasphaera massiliensis strains promote neurite outgrowth in vitro, andmay therefore be used in promoting neuron restoration for the treatmentor prevention of neurodegenerative diseases. Thus, bacterial strains ofthe invention are for use in the treatment or prevention ofneurodegenerative diseases.

The inventors have also found that the bacterial strains of inventionincrease the activation of BDNF. BDNF is a neurotrophic growth factorthat has been shown to enhance neuron differentiation and survival.Thus, the compositions of the invention can be used in a method ofenhancing nerve cell survival in the treatment or prevention ofneurodegenerative diseases.

A further bacteria that may be used in the compositions of the inventionis the species Parabacteroides distasonis. The examples demonstrate thatParabacteroides distasonis and Megasphaera massiliensis both haveneuroprotective activities, but produce different metabolites and mayhave different mechanisms of action and specific neuroprotectiveactivities. Therefore, these species may be particularly effective whenused in combination. In preferred embodiments, the composition comprisesa strain of the species Parabacteroides distasonis and a strain of thespecies Megasphaera massiliensis.

The Parabacteroides distasonis bacterium deposited under accessionnumber NCIMB 42382 was tested in the Examples and is also referred toherein as strain MRx0005. MRX0005, MRX005, MRx005 and MRx0005 are usedherein interchangeably. A 16S rRNA sequence for the MRx0005 strain thatwas tested is provided in SEQ ID NO:17. Strain MRx0005 was depositedwith the international depositary authority NCIMB, Ltd. (FergusonBuilding, Aberdeen, AB21 9YA, Scotland) by GT Biologics Ltd. (LifeSciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) on 12thMarch 2015 as “Parabacteroides sp 755” and was assigned accession numberNCIMB 42382. GT Biologics Ltd. Subsequently changed its name to 4DPharma Research Limited.

In preferred embodiments, the invention provides a compositioncomprising the strain deposited at NCIMB under accession number NCIMB42787, or a derivative or biotype thereof, and the strain deposited atNCIMB under accession number NCIMB 42382, or a derivative or biotypethereof, preferably for use in therapy, preferably for use in treating aneurodegenerative disease such as Parkinson's disease.

Therapeutic Uses

As demonstrated in the examples, the bacterial compositions of theinvention are effective for treating neurodegenerative disorders. Inparticular, treatment with compositions of the invention increaseneuro-proliferation and act as a neuroprotectant against agents thatdestroy dopaminergic neurons. Therefore, the compositions of theinvention may be useful for treating or preventing neurodegenerativedisorders that are the result of neuron death.

Compositions of the invention can decrease the activation of the NFκBpromoter, which activates cytokine production, for example IL-1β, IL-1α,IL-18, TNFα and IL-6. Treating cells with mutant α-synuclein is a modelfor familial Parkinson's. A point mutation at position 53 from adenineto threonine leads to α-synuclein mis-folding. The incorrectly foldedα-synuclein subsequently aggregates into insoluble fibrils which formLewy bodies. Therefore, the compositions of the invention may be usefulfor treating or preventing neurodegenerative disorders that are theresult of neuroinflammation, protein misfolding and/or environmentalexposure. Compositions of the invention can be used for treatment offamilial Parkinson's. Activation of the NFκB promoter is mediatedthrough the TLR4 ligand. TLR4 is known to mediate cell death in themouse model MPTP, which simulates Parkinson's disease. Compositions ofthe invention can be used to inhibit the ability of TLR4 signalling toactivate the NFκB promoter. Of particular relevance for PD, both TLR2and TLR4 were found to be upregulated in brains of PD patients [26].Moreover α-syn has been described as a ligand for TLR2 [27] and we havedemonstrated that α-syn is also a ligand for TLR4 using HEK-TLR4 cells[28].

Compositions of the invention decrease the secretion of pro-inflammatorycytokines such as IL-6, which can be induced by lipopolysaccharide(LPS). Treatment of cells with LPS simulates Parkinson's caused byenvironmental factors. Compositions of the invention can be used todecrease IL-6 secretion. Compositions of the invention can be used fortreatment of environmental Parkinson's.

Examples of neurodegenerative diseases to be treated by compositions ofthe invention include: Parkinson's disease, including progressivesupranuclear palsy, progressive supranuclear palsy,Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus,vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism;Alzheimer's disease, including Benson's syndrome; multiple sclerosis;Huntington's disease; amyotrophic lateral sclerosis; Lou Gehrig'sdisease; motor neurone disease; prion disease; spinocerebellar ataxia;spinal muscular atrophy; dementia, including Lewy body, vascular andfrontotemporal dementia; primary progressive aphasia; mild cognitiveimpairment; HIV-related cognitive impairment, and corticobasaldegeneration. A further disease to be treated by compositions of theinvention is progressive inflammatory neuropathy.

In certain embodiments, the compositions of the invention are for use inreducing neuron death, in particular, in the treatment ofneurodegenerative disorders. In certain embodiments, the compositions ofthe invention are for use in protecting neurons, in particular in thetreatment of neurodegenerative disorders.

In certain embodiments, the compositions of the invention are for use inreducing or preventing loss of dopaminergic cells in the substantianigra. In certain embodiments, the compositions of the invention are foruse in reducing or preventing the degeneration of dopaminergic neuronsin the substantia nigra pars compacta. In certain embodiments, thecompositions of the invention are for use in reducing or preventing thedegeneration of dopaminergic neurons in the substantia nigra parscompacta and the consequent loss of their projecting nerve fibers in thestriatum. In certain embodiments, the compositions of the invention arefor use in reducing or preventing loss of nigrostriatal dopaminergicneurons.

In certain embodiments, the compositions of the invention are for use inincreasing dopamine levels. In certain embodiments, the compositions ofthe invention are for use in increasing DOPAC (3,4-Dihydroxyphenylaceticacid) levels. In certain embodiments, the compositions of the inventionare for use in increasing dopamine and DOPAC levels. In certainembodiments, the dopamine and/or DOPAC levels are increased in thestriatum. Dopamine and DOPAC levels may be measured using anyappropriate method known in the art, such as a radioenzymatic method,for example in plasma or CSF (for example as described in [29]), or areverse-phase HPLC method, perhaps with electrochemical detection, forexample in plasma or CSF (for example as described in [30]).

The neuroprotective properties of the compositions of the invention, asshown in the examples, mean that the compositions may be particularlyeffective for preventing or delaying onset or progression ofneurodegenerative disorders. In certain embodiments, the compositions ofthe invention are for use in delaying onset or progression ofneurodegenerative disorders.

Compositions of the invention can increase the secretion of IL-8. IL-8has been shown to play a role in neuron myelination. In someembodiments, compositions of the invention can be used to increase IL-8secretion.

The therapeutic compositions of the invention can increase theactivation of BDNF. BDNF acts on certain neurons of the central nervoussystem to support the survival of existing neurons and help the growthand development of new neurons and synapses. BDNF is active in thehippocampus, cortex and basal forebrain, and is important for long-termmemory. The compositions of the invention can therefore be used toincrease the secretion of BDNF. The compositions may therefore be usedin the treatment of neurodegenerative diseases associated with theimpairment of long-term memory. The compositions of the invention may beused for improving long-term memory, in particular for improvinglong-term memory that is impaired by a neurodegenerative disease.

In certain embodiments, the compositions of the invention increase themitochondria metabolic activity in neuronal cells.

Modulation of the Microbiota-Gut-Brain Axis

Communication between the gut and the brain (the microbiota-gut-brainaxis) occurs via a bidirectional neurohumoral communication system.Recent evidence shows that the microbiota that resides in the gut canmodulate brain development and produce behavioural phenotypes via themicrobiota-gut-brain axis. Indeed, a number of reviews suggest a role ofthe microbiota-gut-brain axis in maintaining central nervous systemfunctionality and implicate dysfunction of the microbiota-gut-brain axisin the development of central nervous system disorders and conditions[16],[19],[31].

The bidirectional communication between the brain and the gut (i.e.the-gut-brain axis) includes the central nervous system, neuroendocrineand neuroimmune systems, including the hypothalamus-pituitary-adrenal(HPA) axis, sympathetic and parasympathetic arms of the autonomicnervous system (ANS), including the enteric nervous system (ENS) and thevagus nerve, and the gut microbiota.

As demonstrated in the examples, the compositions of the presentinvention can modulate the microbiota-gut-brain axis and reduce celldeath associated with neurodegenerative disorders. Accordingly, thecompositions of the invention may be useful for treating or preventingneurodegenerative disorders, in particular those disorders andconditions associated with dysfunction of the microbiota-gut-brain axis.

In particular embodiments, the compositions of the invention may beuseful for treating or preventing a disease or condition selected fromthe group consisting of: Parkinson's disease, including progressivesupranuclear palsy, progressive supranuclear palsy,Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus,vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism;Alzheimer's disease, including Benson's syndrome; multiple sclerosis;Huntington's disease; amyotrophic lateral sclerosis; Lou Gehrig'sdisease; motor neurone disease; prion disease; spinocerebellar ataxia;spinal muscular atrophy; dementia; including Lewy body; vascular andfrontotemporal dementia; primary progressive aphasia; mild cognitiveimpairment; HIV-related cognitive impairment and corticobasaldegeneration.

The compositions of the invention may be particularly useful fortreating or preventing chronic disease, treating or preventing diseasein patients that have not responded to other therapies (such astreatment with Levodopa, dopamine agonists, MAO-B inhibitors, COMTinhibitors, Glutamate antagonists, and/or anticholinergics), and/ortreating or preventing the tissue damage and symptoms associated withdysfunction of the microbiota-gut-brain axis.

In certain embodiments, the compositions of the invention modulate theCNS. In some embodiments, the compositions of the invention modulate theautonomic nervous system (ANS). In some embodiments, the compositions ofthe invention modulate the enteric nervous system (ENS). In someembodiments, the compositions of the invention modulate thehypothalamic, pituitary, adrenal (HPA) axis. In some embodiments, thecompositions of the invention modulate the neuroendocrine pathway. Insome embodiments, the compositions of the invention modulate theneuroimmune pathway. In some embodiments, the compositions of theinvention modulate the CNS, the ANS, the ENS, the HPA axis and/or theneuroendocrine and neuroimmune pathways. In certain embodiments, thecompositions of the invention module the levels of commensal metabolitesand/or the gastrointestinal permeability of a subject.

The signalling of the microbiota-gut-brain axis is modulated by neuralsystems. Accordingly, in some embodiments, the compositions of theinvention modulate signalling in neural systems. In certain embodiments,the compositions of the invention modulate the signalling of the centralnervous system. In some embodiments, the compositions of the inventionmodulate signalling in sensory neurons. In other embodiments, thecompositions of the invention modulate signalling in motor neurons. Insome embodiments, the compositions of the invention modulate thesignalling in the ANS. In some embodiments, the ANS is theparasympathetic nervous system. In preferred embodiments, thecompositions of the invention modulate the signalling of the vagusnerve. In other embodiments, the ANS is the sympathetic nervous system.In other embodiments, the compositions of the invention modulate thesignalling in the enteric nervous system. In certain embodiments, thesignalling of ANS and ENS neurons responds directly to luminal contentsof the gastrointestinal tract. In other embodiments, the signalling ofANS and ENS neurons responds indirectly to neurochemicals produced byluminal bacteria. In other embodiments, the signalling of ANS and ENSneurons responds to neurochemicals produced by luminal bacteria orenteroendocrine cells. In certain preferred embodiments, the neurons ofthe ENS activate vagal afferents that influence the functions of theCNS. In some embodiments, the compositions of the invention regulate theactivity of enterochromaffin cells.

Neurodegenerative Diseases

Parkinson's Disease

Parkinson's disease is a common neurodegenerative diseaseneuropathologically characterised by degeneration of heterogeneouspopulations of neural cells (dopamine-producing cells). The clinicaldiagnosis of Parkinson's disease requires bradykinesia and at least oneof the following core symptoms: resting tremor; muscle rigidity andpostural reflex impairment. Other signs and symptoms that may be presentor develop during the progression of the disease are autonomicdisturbances (sialorrhoea, seborrhoea, constipation, micturitiondisturbances, sexual functioning, orthostatic hypotension,hyperhydrosis), sleep disturbances and disturbances in the sense ofsmell or sense of temperature. Parkinson's disease is aneurodegenerative disease that may develop or persist due to dysfunctionof the microbiota-gut-brain axis. Therefore, in preferred embodiments,the compositions of the invention are for use in treating or preventingParkinson's disease in a subject.

In further preferred embodiments, the invention provides a compositioncomprising a bacterial strain of the genus Megasphaera, for use in amethod of treating or preventing Parkinson's disease. Compositionscomprising a bacterial strain of the genus Megasphaera may improve motorand cognitive functions in models of Parkinson's disease. Treatment withMegasphaera strains may modulate signalling in the central, autonomicand enteric nervous systems; may modulate the activity of the HPA axispathway; may modulate neuroendocrine and/or neuroimmune pathways; andmay modulate the levels of commensal metabolites, inflammatory markersand/or gastrointestinal permeability of a subject, all of which areimplicated in the neuropathology of Parkinson's disease. In preferredembodiments, the invention provides a composition comprising a bacterialstrain of the species Megasphaera massiliensis for use in a method oftreating or preventing Parkinson's disease. Compositions usingMegasphaera massiliensis may be particularly effective for treatingParkinson's disease.

In preferred embodiments, the compositions of the invention prevent,reduce or alleviate one or more of the symptoms of Parkinson's diseasein a subject. In preferred embodiments, the compositions of theinvention prevent, reduce or alleviate one or more core symptoms ofParkinson's disease in a subject. In certain embodiments, thecompositions of the invention prevent, reduce or alleviate bradykinesiain a subject. In certain embodiments, the compositions of the inventionprevent, reduce or alleviate resting tremor; muscle rigidity and/orpostural reflex impairment in a subject. In certain embodiments, thecompositions of the invention prevent, reduce or alleviate one or moresymptoms associated with Parkinson's disease progression selected fromautonomic disturbances (sialorrhoea, seborrhoea, constipation,micturition disturbances, sexual functioning, orthostatic hypotension,hyperhydrosis), sleep disturbances and disturbances in the sense ofsmell or sense of temperature.

In preferred embodiments, the compositions of the invention prevent,reduce or alleviate depressive symptoms comorbid with Parkinson'sdisease. In certain embodiments, the compositions of the inventionimprove verbal memory and/or executive functions. In certainembodiments, the compositions of the invention improve attention,working memory, verbal fluency and/or anxiety.

In other preferred embodiments, the compositions of the inventionprevent, reduce or alleviate cognitive dysfunctions comorbid withParkinson's disease.

In certain embodiments, the compositions of the invention prevent,reduce or alleviate Parkinson's disease progression. In certainembodiments, the compositions of the invention prevent, reduce oralleviate later motor complications. In certain embodiments, thecompositions of the invention prevent, reduce or alleviate late motorfluctuations. In certain embodiments, the compositions of the inventionprevent, reduce or alleviate neuronal loss. In certain embodiments, thecompositions of the invention improve symptoms of Parkinson's diseasedementia (PDD). In certain embodiments, the compositions of theinvention prevent, reduce or alleviate impairment of executive function,attention and/or working memory. In certain embodiments, thecompositions of the invention improve dopaminergic neurotransmission. Incertain embodiments, the compositions of the invention prevent, reduceor alleviate impaired dopaminergic neurotransmission.

In some embodiments, the compositions of the invention improve thesymptoms of Parkinson's disease according to a symptomatic or diagnosticscale. In certain embodiments, the tests for assessing symptomaticimprovement of motor function in Parkinson's disease is the UnifiedParkinson's Disease Rating Scale. In particular, UPDRS II considers theactivity of daily life and UPDRS III considers motor-examination.

In some embodiments, the compositions of the invention improve thesymptoms associated with PDD according to a symptomatic or diagnostictest and/or scale. In certain embodiments, the test or scale is selectedfrom the Hopkins Verbal Learning Test-Revised (HVLT-R); the Delis-KaplanExecutive Function System (D-KEFS) Color-Word Interference Test; theHamilton Depression Rating Scale (HAM-D 17; depression); the HamiltonAnxiety Rating Scale (HAM-A; anxiety) and the Unified Parkinson'sDisease Rating Scale (UPDRS; PD symptom severity).

In some embodiments, the compositions of the invention improve theClinical Global Impression-Global Improvement (CGI-I) scale forassessing psychiatric and neurological disorders. In some embodiments,the compositions of the invention display a positive effect on globalsocial and occupational impairment of the subject with Parkinson'sdisease.

In certain embodiments, the compositions of the invention are for use intreating or preventing neurological disorders such as Parkinson'sdisease in a subject wherein said use involves reducing or preventingloss of dopaminergic cells in the substantia nigra. In certainembodiments, the compositions of the invention are for use in treatingor preventing neurological disorders such as Parkinson's disease in asubject wherein said use involves reducing or preventing thedegeneration of dopaminergic neurons in the substantia nigra parscompacta. In certain embodiments, the compositions of the invention arefor use in treating or preventing neurological disorders such asParkinson's disease in a subject wherein said use involves reducing orpreventing the degeneration of dopaminergic neurons in the substantianigra pars compacta and the consequent loss of their projecting nervefibers in the striatum. In certain embodiments, the compositions of theinvention are for use in treating or preventing neurological disorderssuch as Parkinson's disease in a subject wherein said use involvesreducing or preventing loss of nigrostriatal dopaminergic neurons.

In certain embodiments, the compositions of the invention are for use intreating or preventing neurological disorders such as Parkinson'sdisease in a subject wherein said use involves increasing dopaminelevels. In certain embodiments, the compositions of the invention arefor use in treating or preventing neurological disorders such asParkinson's disease in a subject wherein said use involves increasingDOPAC levels. In certain embodiments, the compositions of the inventionare for use in treating or preventing neurological disorders such asParkinson's disease in a subject wherein said use involves increasingdopamine and DOPAC levels. In certain embodiments, the dopamine and/orDOPAC levels are increased in the striatum.

Alzheimer's Disease and Dementia

In DSM-5, the term dementia was replaced with the terms majorneurocognitive disorder and mild neurocognitive disorder. Neurocognitivedisorder is a heterogeneous class of psychiatric diseases. The mostcommon neurocognitive disorder is Alzheimer's disease, followed byvascular dementias or mixed forms of the two. Other forms ofneurodegenerative disorders (e.g. Lewy body disease, frontotemporaldementia, Parkinson's dementia, Creutzfeldt-Jakob disease, Huntington'sdisease, and Wernicke-Korsakoff syndrome) are accompanied by dementia.

Alzheimer's disease and dementia are also characterised by neuronalloss, so the neuroprotective and neuroproliferative effects shown in theexamples for the compositions of the invention indicate that they may beuseful for treating or preventing these conditions.

The symptomatic criteria for dementia under DSM-5 are evidence ofsignificant cognitive decline from a previous level of performance inone or more cognitive domains selected from: learning and memory;language; executive function; complex attention; perceptual-motor andsocial cognition. The cognitive deficits must interfere withindependence in everyday activities. In addition, the cognitive deficitsdo not occur exclusively in the context of a delirium and are not betterexplained by another mental disorder (for example MDD or schizophrenia).

In addition to the primary symptom, subjects with neurodegenerativedisorders display behavioural and psychiatric symptoms includingagitation, aggression, depression, anxiety, apathy, psychosis andsleep-wake cycle disturbances.

Neurodegenerative disorders may develop or persist due to dysfunction ofthe microbiota-gut-brain axis. Therefore, in preferred embodiments, thecompositions of the invention are for use in treating or preventingneurodegenerative disorders in a subject. In preferred embodiments, theneurodegenerative disorder is Alzheimer's disease. In other embodiments,the neurodegenerative disorder is selected from vascular dementias;mixed form Alzheimer's disease and vascular dementia; Lewy body disease;frontotemporal dementia; Parkinson's dementia; Creutzfeldt-Jakobdisease; Huntington's disease; and Wernicke-Korsakoff syndrome.

In preferred embodiments, the compositions of the invention prevent,reduce or alleviate one or more of the symptoms of neurodegenerativedisorders in a subject. In certain embodiments, the compositions of theinvention prevent, reduce or alleviate the occurrence of cognitivedecline in a subject. In certain embodiments, the compositions of theinvention improve the level of performance of a subject withneurodegenerative disorders in one or more cognitive domains selectedfrom: learning and memory; language; executive function; complexattention; perceptual-motor and social cognition. In some embodiments,the compositions of the invention prevent, reduce or alleviate theoccurrence of one or more behavioural and psychiatric symptomsassociated with neurodegenerative disorders selected from agitation,aggression, depression, anxiety, apathy, psychosis and sleep-wake cycledisturbances.

In certain embodiments, the compositions of the invention prevent,reduce or alleviate symptomatic disease by intervention in suspectedpathogenic mechanisms at a preclinical stage. In certain embodiments,the compositions of the invention improve disease modification, withslowing or arrest of symptom progression. In some embodiments, theslowing or arrest of symptom progression correlates with evidence indelaying the underlying neuropathological process. In preferredembodiments, the compositions of the invention improve symptoms ofneurodegenerative disorders comprising enhanced cognitive and functionalimprovement. In preferred embodiments, the compositions of the inventionimprove the behavioural and psychiatric symptoms of dementia (BPSD). Inpreferred embodiments, the compositions of the invention improve theability of a subject with neurodegenerative disorder to undertakeeveryday activities.

In preferred embodiments, the compositions of the invention improve bothcognition and functioning in a subject with Alzheimer's disease. In someembodiments, the composition of the invention improves the cognitiveendpoint in a subject with Alzheimer's disease. In some embodiments, thecompositions of the invention improve the functional endpoint in asubject with Alzheimer's disease. In preferred embodiments, thecompositions of the invention improve the cognitive and functionalendpoint in a subject with Alzheimer's disease. In yet further preferredembodiments, the compositions of the invention improve the overallclinical response (the global endpoint) in a subject with Alzheimer'sdisease.

In some embodiments, the compositions of the invention improve thesymptoms of neurodegenerative disorders according to a symptomatic ordiagnostic test. In certain embodiments, the tests for assessingsymptomatic improvement of Alzheimer's disease (and otherneurodegenerative disorders) are selected from objective cognitive,activities of daily living, global assessment of change, health relatedquality of life tests and tests assessing behavioural and psychiatricsymptoms of neurodegenerative disorders.

In certain embodiments, the objective cognitive tests for assessment ofsymptomatic improvement use the Alzheimer's disease Assessment Scalecognitive subscale (ADAS-cog) and the classic ADAS scale. In certainembodiments, symptomatic improvement of cognition is assessed using theNeurophysiological Test Battery for Use in Alzheimer's Disease (NTB).

In some embodiments, the global assessment of change test uses theClinical Global Impression-Global Improvement (CGI-I) scale forassessing psychiatric and neurological disorders. In some embodiments,the global scale is the Clinician's Interview Based Impression of Changeplus (CIBIC-plus). In some embodiments, the global scale is theAlzheimer's Disease Cooperative Study Unit Clinician's Global Impressionof Change (ADCS-CGIC).

In certain embodiments, the health-related quality of life measures arethe Alzheimer's Disease-Related QOL (ADRQL) and the QOL-Alzheimer'sDisease (QOL-AD).

In certain embodiments, the tests assessing behavioural and psychiatricsymptoms of neurodegenerative disorders are selected from theBehavioural pathology in Alzheimer's Disease Rating Scale (BEHAVE-AD);the Behavioural Rating Scale for Dementia (BRSD); the NeuropsychiatricInventory (NPI); and the Cohen-Mansfield Agitation Inventory (CMAI).

In some embodiments, the compositions of the invention are particularlyeffective at preventing, reducing or alleviating neurodegenerativedisorders when used in combination with another therapy for treatingneurodegenerative disorders. In certain embodiments, such therapiesinclude acetylcholinesterase inhibitors including donepezil (Aricept®),galantamine (Razadyne®) and rivastigmine (Exelon®), and memantine.

Multiple Sclerosis

Multiple sclerosis (MS) is a demyelinating disease in which the myelinsheath surrounding neurons in the brain and spinal cord are damaged. Theexact underlying causes of MS are unknown, but are thought to varybetween individuals. Certain forms of MS are hereditary. Environmentalfactors are also thought to contribute to MS. In some individuals, acombination of both genetic and environmental factors may trigger theonset of MS.

There are a wide variety of symptoms associated with MS. Subjects mayexhibit almost any neurological symptom associated with the impairmentof autonomic, visual, motor or sensory control. The exact symptoms willvary depending on the site of neuronal damage/demyelination.

IL-8 has been implicated in the formation of myelin sheaths. Thecompositions of the invention may therefore be for use in theremyelination of neurons in subjects with MS. The compositions of theinvention may also be used to protect neurons from demyelination. Inother words, the compositions of the invention may be for use in amethod of treating or preventing multiple sclerosis by restoring orpreventing loss of neuron myelin sheaths.

In some embodiments, the compositions of the invention prevent, reduceor alleviate one or more symptoms of MS in a subject. In someembodiments, the compositions of the invention prevent, reduce oralleviate fatigue in a subject. In certain embodiments, the compositionsof the invention prevent, reduce or alleviate resting tremor, muscleweakness, muscle spasms, muscle stiffness, paraesthesia and/or ataxia ina subject. In certain embodiments, the compositions of the inventionprevent, reduce or alleviate one or more symptoms associated with MSprogression selected from the list consisting of autonomic disturbances:constipation, micturition disturbances, sexual functioning, dysphagia,dysarthria, syncope, vertigo and/or dizziness; sleep disturbances; anddisturbances in the sense of smell or sense of temperature. In someembodiments, the compositions of the invention prevent, reduce oralleviate one or more ocular symptoms associated with MS. In someembodiments, the ocular symptom is selected from the list consisting ofloss of vision, eye pain, colour blindness, double vision and/orinvoluntary eye movements in a subject.

In some embodiments, the compositions of the invention prevent, reduceor alleviate dizziness, vertigo, neuropathic pain, musculoskeletal pain,cognitive dysfunction, bowel incontinence, dysphagia, dysarthria, or anycombination thereof.

In some embodiments, the compositions of the invention prevent, reduceor alleviate depressive symptoms or anxiety comorbid with MS.

In some embodiments, the improvement of symptoms are determined usingthe 2017 McDonald criteria for diagnosing MS.

In certain embodiments, treatment with the compositions of the inventionresults in a reduction in MS incidence or MS severity. In certainembodiments, the compositions of the invention are for use in reducingrelapse incidence or relapse severity. In certain embodiments, treatmentwith the compositions of the invention prevents a decline in motorfunction or results in improved motor function associated with MS. Incertain embodiments, the compositions of the invention are for use inpreventing a decline in motor function or for use in improving motorfunction in the treatment of MS. In certain embodiments, treatment withthe compositions of the invention prevents the development of paralysisin MS. In certain embodiments, the compositions of the invention are foruse in preventing paralysis in the treatment of MS.

In certain embodiments the compositions of the invention are for use inpreventing multiple sclerosis in a patient that has been identified asat risk of multiple sclerosis, or that has been diagnosed withearly-stage multiple sclerosis or “relapsing-remitting” multiplesclerosis. The compositions of the invention may be useful forpreventing the development of MS. The compositions of the invention maybe useful for preventing the progression of MS. In certain embodiments,the compositions of the invention are for use in a patient identified ashaving a genetic predisposition to MS, such as major histocompatibilitycomplex (MHC) class II phenotype, human leukocyte antigen (HLA)-DR2 orHLA-DR4.

The compositions of the invention may be useful for managing oralleviating multiple sclerosis. The compositions of the invention may beparticularly useful for reducing symptoms associated with multiplesclerosis. Treatment or prevention of multiple sclerosis may refer to,for example, an alleviation of the severity of symptoms or a reductionin the frequency of exacerbations or the range of triggers that are aproblem for the patient. In certain embodiments, the compositions of theinvention slow or stop progression of the disease.

In certain embodiments, the compositions of the invention are for use intreating relapsing-remitting MS. In alternative embodiments, thecompositions of the invention are for use in treating progressive MS,such as secondary progressive MS (SPMS), which develops over timefollowing diagnosis of RRMS, primary progressive MS (PPMS) whichexhibits gradual continuous neurologic deterioration and progressiverelapsing MS (PRMS), which is similar to PPMS but with overlappingrelapses.

In certain embodiments, the compositions of the invention are for use intreating one or more of symptoms of MS selected from the groupconsisting of: fatigue, vision problems, numbness, tingling, musclespasms, muscle stiffness, muscle weakness, mobility problems, pain,problems with thinking, learning and planning, depression and anxiety,sexual problems, bladder problems, bowel problems, speech and swallowingdifficulties.

Neurochemical Factors, Neuropeptides and Neurotransmitters and theMicrobiota-Gut-Brain Axis

As outlined above, the microbiota-gut-brain axis is modulated by anumber of different physiological systems. The microbiota-gut-brain axisis modulated by a number of signalling molecules. Alterations in thelevels of these signalling molecules results in neurodegenerativediseases. The experiments performed by the inventors indicate thatadministration of Megasphaera species, and in particular Megasphaeramassiliensis, can modulate levels of indole and kynurenine.Dysregulation of these metabolites can lead to neurodegenerativediseases, such as Parkinson's disease.

In certain embodiments, the compositions of the invention modulate thelevels of brain monoamines and metabolites thereof. In preferredembodiments the metabolite is kynurenine. In certain embodiments, thecompositions of the invention modulate kynurenine, which is the mainroute of tryptophan metabolism, which serves as a route to nicotinamideadenine dinucleotide (NAD+) production. Kynurenine can be metabolized toneuroactive compounds such as kynurenic acid (KYNA) and3-hydroxy-1-kynurenine (3-OH-1-KYN), and in further steps to quinolinicacid (QUIN). Dysregulation of the kynurenine pathway can lead toactivation of the immune system and the accumulation of potentiallyneurotoxic compounds. Alterations in the kynurenine metabolism may beinvolved in the development of Parkinson's diseases. Kynurenine levelshave been demonstrated to be decreased in the frontal cortex, putamenand substantia nigra pars compacta of patients with PD [32]. Therefore,in certain embodiments the compositions of the invention are for use inincreasing the levels of kynurenine in the treatment of Parkinson'sdisease.

In certain embodiments of the invention the compositions of theinvention can increase the levels kynurenin. Increased levels ofkynurenine have been shown to attenuated MPP+-induced neuronal celldeath in vitro in a human dopaminergic neuroblastoma cell line [33]. Incertain embodiments kynurenine and kynurenic acid, can activate GI arylhydrocarbon receptor (Ahr) and GPR35 receptors. Activation of Ahrreceptor induces IL-22 production, which can inhibit local inflammation.Activation of GPR35 inducing the production of inositol triphosphate andCa2+ mobilization.

In certain embodiments, the compositions of the invention modulate thelevels of indole. In preferred embodiments the metabolite is kynurenine.In certain embodiments, the compositions of the invention modulatekynurenine, which is the main route of tryptophan metabolism.

The signalling of the microbiota-gut-brain axis is modulated by levelsof neurochemical factors, neuropeptides and neurotransmitters.Accordingly, in certain embodiments, the compositions of the inventionmodulates levels of neurochemical factors, neuropeptides andneurotransmitters. Accordingly, in certain preferred embodiments, thecompositions of the invention directly alter CNS biochemistry.

The signalling of the microbiota-gut-brain axis is modulated by levelsof γ-aminobutyric acid (GABA). Accordingly, in preferred embodiments,the compositions of the invention modulate the levels of GABA. GABA isan inhibitory neurotransmitter that reduces neuronal excitability. Incertain embodiments, the compositions of the invention increase thelevels of GABA. In certain embodiments, the compositions of theinvention decrease the levels of GABA. In certain embodiments, thecompositions of the invention alter GABAergic neurotransmission. Incertain embodiments, the compositions of the invention modulate thelevel of GABA transcription in different regions of the central nervoussystem. In certain embodiments, the commensal derived GABA crosses theblood-brain barrier and affects neurotransmission directly. In certainembodiments, the compositions of the invention lead to a reduction ofGABA in the hippocampus, amygdala and/or locus coeruleus. In certainembodiments, the compositions of the invention lead to an increase ofGABA in cortical regions.

Immune Response

The signalling of the microbiota-gut-brain axis is modulated byalterations in the immune response and inflammatory factors and markers.Accordingly, in certain embodiments, the compositions of the inventionmay modulate the immune response. In certain embodiments, thecompositions of the invention modulate the systemic levels ofcirculating neuroimmune signalling molecules. In certain preferredembodiments, the compositions of the invention modulate pro-inflammatorycytokine production and inflammation. In certain embodiments, thecompositions of the invention modulate the inflammatory state. Incertain embodiments, the compositions of the invention decrease IL-6production and secretion. In certain embodiments, the compositions ofthe invention decrease the activation of the NFκB promoter. In certainembodiments, the compositions of the invention are able to modulate theactivation of IL-6 production by the potent pro-inflammatory endotoxinlipopolysaccharide (LPS). In certain embodiments, the compositions ofthe invention are able to modulate the activation of the NFκB promoterby LPS and α-synuclein mutant proteins such as A53T. Increasedcirculating levels of cytokines are closely associated with variousneurodegenerative disorders, including Parkinson's, dementia andAlzheimer's. In certain embodiments, the compositions of the inventionare for use in reducing IL-6 levels and/or NFκB levels in the treatmentof a neurodegenerative disorder.

In some embodiments, the compositions of the invention increase thesecretion of IL-8. IL-8 has been shown to induce myelin sheath formationand restore or preserve effective neuronal communication. Thus, in someembodiments, the compositions of the invention are for use in inducingmyelin sheath formation in the treatment of neurodegenerative diseases.In some embodiments, the compositions of the invention are for use inrestoring neuronal communication. In some embodiments, the compositionsof the invention are for use in preserving neuronal communication.

The signalling of the microbiota-gut-brain axis is modulated by levelsof commensal metabolites. Accordingly, in certain embodiments, thecompositions of the invention modulate the systemic levels of microbiotametabolites. In certain preferred embodiments, the compositions of theinvention modulate the level of short chain fatty acids (SCFAs). Incertain embodiments the level of SCFAs is increased or decreased. Insome embodiments, the SCFA is butyric acid (BA) (or butyrate). In someembodiments, the SCFA is propionic acid (PPA). In some embodiments, theSCFA is acetic acid. In certain embodiments, the compositions of theinvention modulate the ability of SCFAs to cross the blood-brainbarrier.

Histone acetylation and deacetylation are important epigeneticregulators of gene expression. An imbalance in histone acetylation anddeacetylation can result in apoptosis. Dysregulation of such histoneacetyltransferases has been implicated in the pathogenesis associatedwith age-associated neurodegenerative diseases, such as Parkinson'sdisease, Huntington's disease, Alzheimer's disease, amyotrophic lateralsclerosis and cognitive decline [34]. Accordingly, in certainembodiments, the compositions of the invention can modulate histonedeacetylase activity. In certain embodiments, the compositions of theinvention can reduce histone deacetylase activity. In certainembodiments, the compositions of the invention can reduce histoneacetylase activity.

Patients with neurodegenerative diseases, including Parkinson's disease,Huntington's disease, Alzheimer's disease and amyotrophic lateralsclerosis, exhibit high levels of lipid peroxidation. Lipid arevulnerable to oxidation by reactive oxygen species, and the brain isrich in polyunsaturated fatty acids. Accordingly, in certainembodiments, the compositions of the invention can modulate lipidperoxidation. In certain embodiments, the compositions of the inventioncan reduce lipid peroxidation. Reducing the oxidative damage caused byreactive oxygen species can be used to target early the stagesneurodegenerative diseases. Accordingly, in certain embodiments, thecompositions of the invention are for use in treating early stageneurodegeneration. Also accordingly, in certain embodiments, thecompositions of the invention are for use in preventing the developmentof a neurodegenerative disorder. In such embodiments, the compositionsof the invention may be for use in a patient that has been identified asat risk of developing a neurodegenerative disorder.

The signalling of the microbiota-gut-brain axis is modulated by levelsof gastrointestinal permeability. Accordingly, in some embodiments, thecompositions of the invention alter the integrity of thegastrointestinal tract epithelium. In certain embodiments, thecompositions of the invention modulate the permeability of thegastrointestinal tract. In certain embodiments, the compositions of theinvention modulate the barrier function and integrity of thegastrointestinal tract. In certain embodiments, the compositions of theinvention modulate gastrointestinal tract motility. In certainembodiments, the compositions of the invention modulate thetranslocation of commensal metabolites and inflammatory signallingmolecules into the bloodstream from the gastrointestinal tract lumen.

The signalling of the microbiota-gut-brain axis is modulated bymicrobiome composition in the gastrointestinal tract. Accordingly, incertain embodiments, the compositions of the invention modulates themicrobiome composition of the gastrointestinal tract. In certainembodiments, the compositions of the invention prevents microbiomedysbiosis and associated increases in toxic metabolites (e.g. LPS). Incertain embodiments, the compositions of the invention modulate thelevels of Clostridium in the gastrointestinal tract. In preferredembodiments, the compositions of the invention reduce the level ofClostridium in the gastrointestinal tract. In certain embodiments, thecompositions of the invention reduce the levels of Campylobacter jejuni.In certain embodiments, the compositions of the invention modulate theproliferation of harmful anaerobic bacteria and the production ofneurotoxins produced by these bacteria. In certain embodiments, thecompositions of the invention modulate the microbiome levels ofLactobacillus and/or Bifidobacterium. In certain embodiments, thecompositions of the invention modulate the microbiome levels ofSutterella, Prevotella, Ruminococcus genera and/or the Alcaligenaceaefamily. In certain embodiments, the compositions of the inventionincrease the level of Lactobacillus plantarum and/or Saccharomycesboulardii.

Brain Injury

The examples demonstrate that the compositions of the invention areneuroprotective and have HDAC inhibitory activity. HDAC2 is a crucialtarget for functional recovery from stroke [35] and HDAC inhibition canprevent white matter injury [36], so the compositions of the inventionmay be useful in the treatment of brain injury.

In certain embodiments, the compositions of the invention are for use intreating brain injury. In some embodiments, the brain injury is atraumatic brain injury. In some embodiments, the brain injury is anacquired brain injury. In some embodiments, the compositions of theinvention are for use in treating brain injury resulting from trauma. Insome embodiments, the compositions of the invention are for use intreating brain injury resulting from a tumour. In some embodiments, thecompositions of the invention are for use in treating brain injuryresulting from a stroke. In some embodiments, the compositions of theinvention are for use in treating brain injury resulting from a brainhaemorrhage. In some embodiments, the compositions of the invention arefor use in treating brain injury resulting from encephalitis. In someembodiments, the compositions of the invention are for use in treatingbrain injury resulting from cerebral hypoxia. In some embodiments, thecompositions of the invention are for use in treating brain injuryresulting from cerebral anoxia.

In preferred embodiments, the compositions of the invention are for usein treating stroke. The effects shown in the examples are particularlyrelevant to the treatment of stroke. Stroke occurs when blood flow to atleast a part of the brain is interrupted. Without an adequate supply ofblood to provide oxygen and nutrients to the brain tissue and to removewaste products from the brain tissue, brain cells rapidly begin to die.The symptoms of stroke are dependent on the region of the brain which isaffected by the inadequate blood flow. Symptoms include paralysis,numbness or weakness of the muscles, loss of balance, dizziness, suddensevere headaches, speech impairment, loss of memory, loss of reasoningability, sudden confusion, vision impairment, coma or even death. Astroke is also referred to as a brain attack or a cerebrovascularaccident (CVA). The symptoms of stroke may be brief if adequate bloodflow is restored within a short period of time. However, if inadequateblood flow continues for a significant period of time, the symptoms canbe permanent.

In some embodiments, the stroke is cerebral ischemia. Cerebral ischemiaresults when there is insufficient blood flow to the tissues of thebrain to meet metabolic demand. In some embodiments, the cerebralischemia is focal cerebral ischemia, i.e. confined to a specific regionof the brain. In some embodiments the cerebral ischemia is globalcerebral ischemia, i.e. encompassing a wide area of the brain tissue.Focal cerebral ischemia commonly occurs when a cerebral vessel hasbecome blocked, either partially or completely, reducing the flow ofblood to a specific region of the brain. In some embodiments the focalcerebral ischemia is ischemic stroke. In some embodiments, the ischemicstroke is thrombotic, i.e. caused by a thrombus or blood clot, whichdevelops in a cerebral vessel and restricts or blocks blood flow. Insome embodiments the ischemic stroke is a thrombotic stroke. In someembodiments, the ischemic stroke is embolic, i.e. caused by an embolus,or an unattached mass that travels through the bloodstream and restrictsor blocks blood flow at a site distant from its point of origin. In someembodiments the ischemic stroke is an embolic stroke. Global cerebralischemia commonly occurs when blood flow to the brain as a whole isblocked or reduced. In some embodiments the global cerebral ischemia iscaused by hypoperfusion, i.e. due to shock. In some embodiments theglobal cerebral ischemia is a result of a cardiac arrest.

In some embodiments the subject diagnosed with brain injury has sufferedcerebral ischemia. In some embodiments, the subject diagnosed with braininjury has suffered focal cerebral ischemia. In some embodiments, thesubject diagnosed with brain injury has suffered an ischemic stroke. Insome embodiments, the subject diagnosed with brain injury has suffered athrombotic stroke. In some embodiments, the subject diagnosed with braininjury has suffered an embolic stroke. In some embodiments, the subjectdiagnosed with brain injury has suffered global cerebral ischemia. Insome embodiments, the subject diagnosed with brain injury has sufferedhypoperfusion. In some embodiments, the subject diagnosed with braininjury has suffered a cardiac arrest.

In some embodiments, the compositions of the invention are for use intreating cerebral ischemia. In some embodiments, the compositions of theinvention are for use in treating focal cerebral ischemia. In someembodiments, the compositions of the invention are for use treatingischemic stroke. In some embodiments, the compositions of the inventionare for use in treating thrombotic stroke. In some embodiments, thecompositions of the invention are for use in treating embolic stroke. Insome embodiments, the compositions of the invention are for use intreating global cerebral ischemia. In some embodiments, the compositionsof the invention are for use in treating hypoperfusion.

In some embodiments, the stroke is hemorrhagic stroke. Hemorrhagicstroke is caused by bleeding into or around the brain resulting inswelling, pressure and damage to the cells and tissues of the brain.Hemorrhagic stroke is commonly a result of a weakened blood vessel thatruptures and bleeds into the surrounding brain. In some embodiments, thehemorrhagic stroke is an intracerebral hemorrhage, i.e. caused bybleeding within the brain tissue itself. In some embodiments theintracerebral hemorrhage is caused by an intraparenchymal hemorrhage. Insome embodiments the intracerebral hemorrhage is caused by anintraventricular hemorrhage. In some embodiments the hemorrhagic strokeis a subarachnoid hemorrhage i.e. bleeding that occurs outside of thebrain tissue but still within the skull. In some embodiments, thehemorrhagic stroke is a result of cerebral amyloid angiopathy. In someembodiments, the hemorrhagic stroke is a result of a brain aneurysm. Insome embodiments, the hemorrhagic stroke is a result of cerebralarteriovenous malformation (AVM).

In some embodiments the subject diagnosed with brain injury has sufferedhemorrhagic stroke. In some embodiments, the subject diagnosed withbrain injury has suffered an intracerebral hemorrhage. In someembodiments, the subject diagnosed with brain injury has suffered anintraparenchymal hemorrhage. In some embodiments, the subject diagnosedwith brain injury has suffered an intraventricular hemorrhage. In someembodiments, the subject diagnosed with brain injury has suffered asubarachnoid hemorrhage. In some embodiments, the subject diagnosed withbrain injury has suffered cerebral amyloid angiopathy. In someembodiments, the subject diagnosed with brain injury has suffered abrain aneurysm. In some embodiments, the subject diagnosed with braininjury has suffered cerebral AVM.

In some embodiments, the compositions of the invention are for use intreating hemorrhagic stroke. In some embodiments, the compositions ofthe invention are for use in treating an intracerebral hemorrhage. Insome embodiments, the compositions of the invention are for use intreating an intraparenchymal hemorrhage. In some embodiments, thecompositions of the invention are for use in treating anintraventricular hemorrhage. In some embodiments, the compositions ofthe invention are for use in treating a subarachnoid hemorrhage. In someembodiments, the compositions of the invention are for use in treating acerebral amyloid angiopathy. In some embodiments, the compositions ofthe invention are for use in treating a brain aneurysm. In someembodiments, the compositions of the invention are for use in treatingcerebral AVM.

Restoration of adequate blood flow to the brain after a period ofinterruption, though effective in alleviating the symptoms associatedwith stroke, can paradoxically result in further damage to the braintissue. During the period of interruption, the affected tissue suffersfrom a lack of oxygen and nutrients, and the sudden restoration of bloodflow can result in inflammation and oxidative damage through theinduction of oxidative stress. This is known as reperfusion injury, andis well documented not only following stroke, but also following a heartattack or other tissue damage when blood supply returns to the tissueafter a period of ischemia or lack of oxygen. In some embodiments thesubject diagnosed with brain injury has suffered from reperfusion injuryas a result of stroke. In some embodiments, the compositions of theinvention are for use in treating reperfusion injury as a result ofstroke.

A transient ischemic attack (TIA), often referred to as a mini-stroke,is a recognised warning sign for a more serious stroke. Subjects whohave suffered one or more TIAs are therefore at greater risk of stroke.In some embodiments the subject diagnosed with brain injury has suffereda TIA. In some embodiments, the compositions of the invention are foruse in treating a TIA. In some embodiments, the compositions of theinvention are for use in treating brain injury in a subject who hassuffered a TIA.

High blood pressure, high blood cholesterol, a familial history ofstroke, heart disease, diabetes, brain aneurysms, arteriovenousmalformations, sickle cell disease, vasculitis, bleeding disorders, useof nonsteroidal anti-inflammatory drugs (NSAIDs), smoking tobacco,drinking large amounts of alcohol, illegal drug use, obesity, lack ofphysical activity and an unhealthy diet are all considered to be riskfactors for stroke. In particular, lowering blood pressure has beenconclusively shown to prevent both ischemic and hemorrhagic strokes [37,38]. In some embodiments, the compositions of the invention are for usein treating brain injury in a subject who has at least one risk factorfor stroke. In some embodiments the subject has two risk factors forstroke. In some embodiments the subject has three risk factors forstroke. In some embodiments the subject has four risk factors forstroke. In some embodiments the subject has more than four risk factorsfor stroke. In some embodiments the subject has high blood pressure. Insome embodiments the subject has high blood cholesterol. In someembodiments the subject has a familial history of stroke. In someembodiments the subject has heart disease. In some embodiments thesubject has diabetes. In some embodiments the subject has a brainaneurysm. In some embodiments the subject has arteriovenousmalformations. In some embodiments the subject has vasculitis. In someembodiments the subject has sickle cell disease. In some embodiments thesubject has a bleeding disorder. In some embodiments the subject has ahistory of use of nonsteroidal anti-inflammatory drugs (NSAIDs). In someembodiments the subject smokes tobacco. In some embodiments the subjectdrinks large amounts of alcohol. In some embodiments the subject usesillegal drugs. In some embodiments the subject is obese. In someembodiments the subject is overweight. In some embodiments the subjecthas a lack of physical activity. In some embodiments the subject has anunhealthy diet.

The examples indicate that the compositions of the invention may beuseful for treating brain injury and aiding recovery when administeredbefore the injury event occurs. Therefore, the compositions of theinvention may be particularly useful for treating brain injury whenadministered to subjects at risk of brain injury, such as stroke.

In certain embodiments, the compositions of the invention are for use inreducing the damage caused by a potential brain injury, preferably astroke. The compositions may reduce the damage caused when they areadministered before the potential brain injury occurs, in particularwhen administered to a patient identified as at risk of a brain injury.

The examples indicate that the compositions of the invention may beuseful for treating brain injury and aiding recovery when administeredafter the injury event occurs. Therefore, the compositions of theinvention may be particularly useful for treating brain injury whenadministered to subjects following a brain injury, such as stroke.

In some embodiments, the compositions of the invention treat braininjury by reducing motoric damage. In some embodiments, the compositionsof the invention treat brain injury by improving motor function. In someembodiments, the compositions of the invention treat brain injury byimproving muscle strength. In some embodiments, the compositions of theinvention treat brain injury by improving memory. In some embodiments,the compositions of the invention treat brain injury by improving socialrecognition. In some embodiments, the compositions of the inventiontreat brain injury by improving neurological function.

Treatment of brain injury may refer to, for example, an alleviation ofthe severity of symptoms. Treatment of brain injury may also refer toreducing the neurological impairments following stroke. Compositions ofthe invention for use in treating stroke may be provided to the subjectin advance of the onset of stroke, for example in a patient identifiedas being at risk of stroke. Compositions of the invention for use intreating stroke may be provided after a stroke has occurred, forexample, during recovery. Compositions of the invention for use intreating stroke may be provided during the acute phase of recovery (i.e.up to one week after stroke). Compositions of the invention for use intreating stroke may be provided during the subacute phase of recovery(i.e. from one week up to three months after stroke). Compositions ofthe invention for use in treating stroke may be provided during thechronic phase of recovery (from three months after stroke).

In certain embodiments, the compositions of the invention are for use incombination with a secondary active agent. In certain embodiments, thecompositions of the invention are for use in combination with aspirin ortissue plasminogen activator (tPA). Other secondary agents include otherantiplatelets (such as clopidogrel), anticoagulants (such as heparins,warfarin, apixaban, dabigatran, edoxaban or rivaroxaban),antihypertensives (such as diuretics, ACE inhibitors, calcium channelblockers, beta-blockers or alpha-blockers) or statins. The compositionsof the invention may improve the patient's response to the secondaryactive agent.

In certain embodiments, the compositions of the invention reduce theeffect of ischemia on tissues. In certain embodiments, the compositionsof the invention reduce the amount of damage to tissues caused byischemia. In certain embodiments, the tissues damaged by ischemia arethe cerebral tissues. In certain embodiments, the compositions of theinvention reduce necrosis or the number of necrotic cells. In certainembodiments, the compositions of the invention reduce apoptosis or thenumber of apoptotic cells. In certain embodiments, the compositions ofthe invention reduce the number of necrotic and apoptotic cells. Incertain embodiments, the compositions of the invention prevent celldeath by necrosis and/or apoptosis. In certain embodiments, thecompositions of the invention prevent cell death by necrosis and/orapoptosis caused by ischemia. In certain embodiments, the compositionsof the invention improve the recovery of the tissue damaged by ischemia.In certain embodiments, the compositions of the invention improve thespeed of clearance of necrotic cells and/or apoptotic cells. In certainembodiments, the compositions of the invention improve the efficacy ofthe clearance of necrotic cells and/or apoptotic cells. In certainembodiments, the compositions of the invention improve the replacementand/or regeneration of cells within tissues. In certain embodiments, thecompositions of the invention improve the replacement and/orregeneration of cells within tissues damaged by ischemia. In certainembodiments, the compositions of the invention improve the overallhistology of the tissue (for example upon a biopsy).

Modes of Administration

Preferably, the compositions of the invention are to be administered tothe gastrointestinal tract in order to enable delivery to and/or partialor total colonisation of the intestine with the bacterial strain of theinvention. Generally, the compositions of the invention are administeredorally, but they may be administered rectally, intranasally, or viabuccal or sublingual routes.

In certain embodiments, the compositions of the invention may beadministered as a foam, as a spray or a gel.

In certain embodiments, the compositions of the invention may beadministered as a suppository, such as a rectal suppository, for examplein the form of a theobroma oil (cocoa butter), synthetic hard fat (e.g.suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soapglycerin composition.

In certain embodiments, the composition of the invention is administeredto the gastrointestinal tract via a tube, such as a nasogastric tube,orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneousendoscopic gastrostomy (PEG), or a port, such as a chest wall port thatprovides access to the stomach, jejunum and other suitable access ports.

The compositions of the invention may be administered once, or they maybe administered sequentially as part of a treatment regimen. In certainembodiments, the compositions of the invention are to be administereddaily.

In certain embodiments of the invention, treatment according to theinvention is accompanied by assessment of the patient's gut microbiota.Treatment may be repeated if delivery of and/or partial or totalcolonisation with the strain of the invention is not achieved such thatefficacy is not observed, or treatment may be ceased if delivery and/orpartial or total colonisation is successful and efficacy is observed.

In certain embodiments, the composition of the invention may beadministered to a pregnant animal, for example a mammal such as a humanin order to prevent an inflammatory or autoimmune disease developing inher child in utero and/or after it is born.

The compositions of the invention may be administered to a patient thathas been diagnosed with a neurodegenerative disease, or that has beenidentified as being at risk of a neurodegenerative disease. Thecompositions may also be administered as a prophylactic measure toprevent the development of neurodegenerative disease in a healthypatient.

The compositions of the invention may be administered to a patient thathas been identified as having an abnormal gut microbiota. For example,the patient may have reduced or absent colonisation by Megasphaera, andin particular Megasphaera massiliensis.

The compositions of the invention may be administered as a food product,such as a nutritional supplement.

Generally, the compositions of the invention are for the treatment ofhumans, although they may be used to treat animals including monogastricmammals such as poultry, pigs, cats, dogs, horses or rabbits. Thecompositions of the invention may be useful for enhancing the growth andperformance of animals. If administered to animals, oral gavage may beused.

Compositions

Generally, the composition of the invention comprises bacteria. Inpreferred embodiments of the invention, the composition is formulated infreeze-dried form. For example, the composition of the invention maycomprise granules or gelatin capsules, for example hard gelatincapsules, comprising a bacterial strain of the invention.

Preferably, the composition of the invention comprises lyophilisedbacteria. Lyophilisation of bacteria is a well-established procedure andrelevant guidance is available in, for example, references [39], [ ],[41]].

Alternatively, the composition of the invention may comprise a live,active bacterial culture.

In some embodiments, the bacterial strain in the composition of theinvention has not been inactivated, for example, has not beenheat-inactivated. In some embodiments, the bacterial strain in thecomposition of the invention has not been killed, for example, has notbeen heat-killed. In some embodiments, the bacterial strain in thecomposition of the invention has not been attenuated, for example, hasnot been heat-attenuated. For example, in some embodiments, thebacterial strain in the composition of the invention has not beenkilled, inactivated and/or attenuated. For example, in some embodiments,the bacterial strain in the composition of the invention is live. Forexample, in some embodiments, the bacterial strain in the composition ofthe invention is viable. For example, in some embodiments, the bacterialstrain in the composition of the invention is capable of partially ortotally colonising the intestine. For example, in some embodiments, thebacterial strain in the composition of the invention is viable andcapable of partially or totally colonising the intestine.

In some embodiments, the composition comprises a mixture of livebacterial strains and bacterial strains that have been killed.

In preferred embodiments, the composition of the invention isencapsulated to enable delivery of the bacterial strain to theintestine. Encapsulation protects the composition from degradation untildelivery at the target location through, for example, rupturing withchemical or physical stimuli such as pressure, enzymatic activity, orphysical disintegration, which may be triggered by changes in pH. Anyappropriate encapsulation method may be used. Exemplary encapsulationtechniques include entrapment within a porous matrix, attachment oradsorption on solid carrier surfaces, self-aggregation by flocculationor with cross-linking agents, and mechanical containment behind amicroporous membrane or a microcapsule. Guidance on encapsulation thatmay be useful for preparing compositions of the invention is availablein, for example, references [42] and [43].

The composition may be administered orally and may be in the form of atablet, capsule or powder. Encapsulated products are preferred becauseMegasphaera are anaerobes. Other ingredients (such as vitamin C, forexample), may be included as oxygen scavengers and prebiotic substratesto improve the delivery and/or partial or total colonisation andsurvival in vivo. Alternatively, the probiotic composition of theinvention may be administered orally as a food or nutritional product,such as milk or whey based fermented dairy product, or as apharmaceutical product.

The composition may be formulated as a probiotic.

A composition of the invention includes a therapeutically effectiveamount of a bacterial strain of the invention. A therapeuticallyeffective amount of a bacterial strain is sufficient to exert abeneficial effect upon a patient. A therapeutically effective amount ofa bacterial strain may be sufficient to result in delivery to and/orpartial or total colonisation of the patient's intestine.

A suitable daily dose of the bacteria, for example for an adult human,may be from about 1×10³ to about 1×10¹¹ colony forming units (CFU); forexample, from about 1×10⁷ to about 1×10¹⁰ CFU; in another example fromabout 1×10⁶ to about 1×10¹⁰ CFU.

In certain embodiments, the composition contains the bacterial strain inan amount of from about 1×10⁶ to about 1×10¹¹ CFU/g, respect to theweight of the composition; for example, from about 1×10⁸ to about 1×10¹⁰CFU/g. The dose may be, for example, 1 g, 3 g, 5 g, and 10 g.

Typically, a probiotic, such as the composition of the invention, isoptionally combined with at least one suitable prebiotic compound. Aprebiotic compound is usually a non-digestible carbohydrate such as anoligo- or polysaccharide, or a sugar alcohol, which is not degraded orabsorbed in the upper digestive tract. Known prebiotics includecommercial products such as inulin and transgalacto-oligosaccharides.

In certain embodiments, the probiotic composition of the presentinvention includes a prebiotic compound in an amount of from about 1 toabout 30% by weight, respect to the total weight composition, (e.g. from5 to 20% by weight). Carbohydrates may be selected from the groupconsisting of: fructo-oligosaccharides (or FOS), short-chainfructo-oligosaccharides, inulin, isomalt-oligosaccharides, pectins,xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS),beta-glucans, arable gum modified and resistant starches, polydextrose,D-tagatose, acacia fibers, carob, oats, and citrus fibers. In oneaspect, the prebiotics are the short-chain fructo-oligosaccharides (forsimplicity shown herein below as FOSs-c.c); said FOSs-c.c. are notdigestible carbohydrates, generally obtained by the conversion of thebeet sugar and including a saccharose molecule to which three glucosemolecules are bonded.

In certain embodiments, the compositions of the invention are used incombination with another therapeutic compound for treating or preventingthe neurodegenerative disorder. In some embodiments, the compositions ofthe invention are administered with nutritional supplements thatmodulate neuroprotection or neuroproliferation. In preferredembodiments, the nutritional supplements comprise or consist ofnutritional vitamins. In certain embodiments, the vitamins are vitaminB6, magnesium, dimethylglycine (vitamin B16) and vitamin C. In certainembodiments, the compositions of the invention are administered incombination with another probiotic.

In certain embodiments, the compositions of the invention are for use inenhancing the effect of a second agent on a neurodegenerative disease.The immune modulatory effects of the compositions of the invention maymake the brain more susceptible to conventional therapies such asLevodopa, dopamine agonists, MAO-B inhibitors, COMT inhibitors,Glutamate antagonists, or anticholinergics, which are exemplarysecondary agents to be administered in combination (sequentially orcontemporaneously) with the compositions of the invention.

The compositions of the invention may comprise pharmaceuticallyacceptable excipients or carriers. Examples of such suitable excipientsmay be found in the reference [44]. Acceptable carriers or diluents fortherapeutic use are well known in the pharmaceutical art and aredescribed, for example, in reference [45]. Examples of suitable carriersinclude lactose, starch, glucose, methyl cellulose, magnesium stearate,mannitol, sorbitol and the like. Examples of suitable diluents includeethanol, glycerol and water. The choice of pharmaceutical carrier,excipient or diluent can be selected with regard to the intended routeof administration and standard pharmaceutical practice. Thepharmaceutical compositions may comprise as, or in addition to, thecarrier, excipient or diluent any suitable binder(s), lubricant(s),suspending agent(s), coating agent(s), solubilising agent(s). Examplesof suitable binders include starch, gelatin, natural sugars such asglucose, anhydrous lactose, free-flow lactose, beta-lactose, cornsweeteners, natural and synthetic gums, such as acacia, tragacanth orsodium alginate, carboxymethyl cellulose and polyethylene glycol.Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Preservatives, stabilizers, dyes and even flavouring agentsmay be provided in the pharmaceutical composition. Examples ofpreservatives include sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid. Antioxidants and suspending agents may be alsoused.

The compositions of the invention may be formulated as a food product.For example, a food product may provide nutritional benefit in additionto the therapeutic effect of the invention, such as in a nutritionalsupplement. Similarly, a food product may be formulated to enhance thetaste of the composition of the invention or to make the compositionmore attractive to consume by being more similar to a common food item,rather than to a pharmaceutical composition. In certain embodiments, thecomposition of the invention is formulated as a milk-based product. Theterm “milk-based product” means any liquid or semi-solid milk- orwhey-based product having a varying fat content. The milk-based productcan be, e.g., cow's milk, goat's milk, sheep's milk, skimmed milk, wholemilk, milk recombined from powdered milk and whey without anyprocessing, or a processed product, such as yoghurt, curdled milk, curd,sour milk, sour whole milk, butter milk and other sour milk products.Another important group includes milk beverages, such as whey beverages,fermented milks, condensed milks, infant or baby milks; flavoured milks,ice cream; milk-containing food such as sweets.

In some embodiments, the compositions of the invention comprise one ormore bacterial strains of the genus Megasphaera and do not containbacteria from any other genera, or which comprise only de minimis orbiologically irrelevant amounts of bacteria from another genera. Thus,in some embodiments, the invention provides a composition comprising oneor more bacterial strains of the genus Megasphaera, which does notcontain bacteria from any other genera or which comprises only deminimis or biologically irrelevant amounts of bacteria from anothergenera, for use in therapy.

In some embodiments, the compositions of the invention comprise one ormore bacterial strains of the species Megasphaera massiliensis and donot contain bacteria from any other species, or which comprise only deminimis or biologically irrelevant amounts of bacteria from anotherspecies. Thus, in some embodiments, the invention provides a compositioncomprising one or more bacterial strains of the species Megasphaeramassiliensis, which does not contain bacteria from any other species orwhich comprises only de minimis or biologically irrelevant amounts ofbacteria from another species, for use in therapy.

In some embodiments, the compositions of the invention comprise one ormore bacterial strains of the species Megasphaera massiliensis and donot contain bacteria from any other Megasphaera species, or whichcomprise only de minimis or biologically irrelevant amounts of bacteriafrom another Megasphaera species. Thus, in some embodiments, theinvention provides a composition comprising one or more bacterialstrains of the species Megasphaera massiliensis, which does not containbacteria from any other Megasphaera species or which comprises only deminimis or biologically irrelevant amounts of bacteria from anotherMegasphaera species, for use in therapy.

In certain embodiments, the compositions of the invention contain asingle bacterial strain or species and do not contain any otherbacterial strains or species. Such compositions may comprise only deminimis or biologically irrelevant amounts of other bacterial strains orspecies. Such compositions may be a culture that is substantially freefrom other species of organism.

In some embodiments, the invention provides a composition comprising asingle bacterial strain of the genus Megasphaera, which does not containbacteria from any other strains or which comprises only de minimis orbiologically irrelevant amounts of bacteria from another strain for usein therapy.

In some embodiments, the invention provides a composition comprising asingle bacterial strain of the species Megasphaera massiliensis, whichdoes not contain bacteria from any other strains or which comprises onlyde minimis or biologically irrelevant amounts of bacteria from anotherstrain for use in therapy.

In some embodiments, the compositions of the invention comprise morethan one bacterial strain. For example, in some embodiments, thecompositions of the invention comprise more than one strain from withinthe same species (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40 or 45 strains), and, optionally, do not contain bacteriafrom any other species. In some embodiments, the compositions of theinvention comprise less than 50 strains from within the same species(e.g. less than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4 or3 strains), and, optionally, do not contain bacteria from any otherspecies. In some embodiments, the compositions of the invention comprise1-40, 1-30, 1-20, 1-19, 1-18, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4,1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or31-50 strains from within the same species and, optionally, do notcontain bacteria from any other species. The invention comprises anycombination of the foregoing.

In some embodiments, the composition comprises a microbial consortium.For example, in some embodiments, the composition comprises theMegasphaera bacterial strain as part of a microbial consortium. Forexample, in some embodiments, the Megasphaera bacterial strain ispresent in combination with one or more (e.g. at least 2, 3, 4, 5, 10,15 or 20) other bacterial strains from other genera with which it canlive symbiotically in vivo in the intestine. For example, in someembodiments, the composition comprises a bacterial strain of Megasphaerain combination with a bacterial strain from a different genus. In someembodiments, the microbial consortium comprises two or more bacterialstrains obtained from a faeces sample of a single organism, e.g. ahuman. In some embodiments, the microbial consortium is not foundtogether in nature. For example, in some embodiments, the microbialconsortium comprises bacterial strains obtained from faeces samples ofat least two different organisms. In some embodiments, the two differentorganisms are from the same species, e.g. two different humans. In someembodiments, the two different organisms are an infant human and anadult human. In some embodiments, the two different organisms are ahuman and a non-human mammal.

In some embodiments, the composition of the invention additionallycomprises a bacterial strain that has the same safety and therapeuticefficacy characteristics as strain MRx0029, but which is not MRx0029, orwhich is not a Megasphaera massiliensis.

In some embodiments in which the composition of the invention comprisesmore than one bacterial strain, species or genus, the individualbacterial strains, species or genera may be for separate, simultaneousor sequential administration. For example, the composition may compriseall of the more than one bacterial strain, species or genera, or thebacterial strains, species or genera may be stored separately and beadministered separately, simultaneously or sequentially. In someembodiments, the more than one bacterial strains, species or genera arestored separately but are mixed together prior to use.

In some embodiments, the bacterial strain for use in the invention isobtained from human adult faeces. In some embodiments in which thecomposition of the invention comprises more than one bacterial strain,all of the bacterial strains are obtained from human adult faeces or ifother bacterial strains are present they are present only in de minimisamounts. The bacteria may have been cultured subsequent to beingobtained from the human adult faeces and being used in a composition ofthe invention.

As mentioned above, in some embodiments, the one or more Megasphaerabacterial strains is/are the only therapeutically active agent(s) in acomposition of the invention. In some embodiments, the bacterialstrain(s) in the composition is/are the only therapeutically activeagent(s) in a composition of the invention.

The compositions for use in accordance with the invention may or may notrequire marketing approval.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is lyophilised. In certainembodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is spray dried. In certainembodiments, the invention provides the above pharmaceuticalcomposition, wherein the bacterial strain is lyophilised or spray driedand wherein it is live. In certain embodiments, the invention providesthe above pharmaceutical composition, wherein the bacterial strain islyophilised or spray dried and wherein it is viable. In certainembodiments, the invention provides the above pharmaceuticalcomposition, wherein the bacterial strain is lyophilised or spray driedand wherein it is capable of partially or totally colonising theintestine. In certain embodiments, the invention provides the abovepharmaceutical composition, wherein the bacterial strain is lyophilisedor spray dried and wherein it is viable and capable of partially ortotally colonising the intestine.

In some cases, the lyophilised bacterial strain is reconstituted priorto administration. In some cases, the reconstitution is by use of adiluent described herein.

The compositions of the invention can comprise pharmaceuticallyacceptable excipients, diluents or carriers.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a neurodegenerativedisorder when administered to a subject in need thereof

In certain embodiments, the invention provides pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat or prevent aneurodegenerative disorder.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the amount of the bacterial strain is from about1×10³ to about 1×10¹¹ colony forming units per gram with respect to aweight of the composition.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the composition is administered at a dose of 1 g, 3g, 5 g or 10 g.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the composition is administered by a methodselected from the group consisting of oral, rectal, subcutaneous, nasal,buccal, and sublingual.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a carrier selected from the group consisting oflactose, starch, glucose, methyl cellulose, magnesium stearate, mannitoland sorbitol.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a diluent selected from the group consisting ofethanol, glycerol and water.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising an excipient selected from the group consistingof starch, gelatin, glucose, anhydrous lactose, free-flow lactose,beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate,carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate and sodiumchloride.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, further comprising at least one of a preservative, anantioxidant and a stabilizer.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a preservative selected from the groupconsisting of sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is lyophilised.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein when the composition is stored in a sealedcontainer at about 4.0 or about 25.0 and the container is placed in anatmosphere having 50% relative humidity, at least 80% of the bacterialstrain as measured in colony forming units, remains after a period of atleast about: 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years,2.5 years or 3 years.

In some embodiments, the composition of the invention is provided in asealed container comprising a composition as described herein. In someembodiments, the sealed container is a sachet or bottle. In someembodiments, the composition of the invention is provided in a syringecomprising a composition as described herein.

The composition of the present invention may, in some embodiments, beprovided as a pharmaceutical formulation. For example, the compositionmay be provided as a tablet or capsule. In some embodiments, the capsuleis a gelatine capsule (“gel-cap”).

In some embodiments, the compositions of the invention are administeredorally. Oral administration may involve swallowing, so that the compoundenters the gastrointestinal tract, and/or buccal, lingual, or sublingualadministration by which the compound enters the blood stream directlyfrom the mouth.

Pharmaceutical formulations suitable for oral administration includesolid plugs, solid microparticulates, semi-solid and liquid (includingmultiple phases or dispersed systems) such as tablets; soft or hardcapsules containing multi- or nano-particulates, liquids (e.g. aqueoussolutions), emulsions or powders; lozenges (including liquid-filled);chews; gels; fast dispersing dosage forms; films; ovules; sprays; andbuccal/mucoadhesive patches.

In some embodiments the pharmaceutical formulation is an entericformulation, i.e. a gastro-resistant formulation (for example, resistantto gastric pH) that is suitable for delivery of the composition of theinvention to the intestine by oral administration. Enteric formulationsmay be particularly useful when the bacteria or another component of thecomposition is acid-sensitive, e.g. prone to degradation under gastricconditions.

In some embodiments, the enteric formulation comprises an entericcoating. In some embodiments, the formulation is an enteric-coateddosage form. For example, the formulation may be an enteric-coatedtablet or an enteric-coated capsule, or the like. The enteric coatingmay be a conventional enteric coating, for example, a conventionalcoating for a tablet, capsule, or the like for oral delivery. Theformulation may comprise a film coating, for example, a thin film layerof an enteric polymer, e.g. an acid-insoluble polymer.

In some embodiments, the enteric formulation is intrinsically enteric,for example, gastro-resistant without the need for an enteric coating.Thus, in some embodiments, the formulation is an enteric formulationthat does not comprise an enteric coating. In some embodiments, theformulation is a capsule made from a thermogelling material. In someembodiments, the thermogelling material is a cellulosic material, suchas methylcellulose, hydroxymethylcellulose orhydroxypropylmethylcellulose (HPMC). In some embodiments, the capsulecomprises a shell that does not contain any film forming polymer. Insome embodiments, the capsule comprises a shell and the shell compriseshydroxypropylmethylcellulose and does not comprise any film formingpolymer (e.g. see [46]). In some embodiments, the formulation is anintrinsically enteric capsule (for example, Vcaps® from Capsugel).

In some embodiments, the formulation is a soft capsule. Soft capsulesare capsules which may, owing to additions of softeners, such as, forexample, glycerol, sorbitol, maltitol and polyethylene glycols, presentin the capsule shell, have a certain elasticity and softness. Softcapsules can be produced, for example, on the basis of gelatine orstarch. Gelatine-based soft capsules are commercially available fromvarious suppliers. Depending on the method of administration, such as,for example, orally or rectally, soft capsules can have various shapes,they can be, for example, round, oval, oblong or torpedo-shaped. Softcapsules can be produced by conventional processes, such as, forexample, by the Scherer process, the Accogel process or the droplet orblowing process.

Culturing Methods

The bacterial strains for use in the present invention can be culturedusing standard microbiology techniques as detailed in, for example,references [47], [ ] and [49].

The solid or liquid medium used for culture may be YCFA agar or YCFAmedium. YCFA medium may include (per 100 ml, approximate values):Casitone (1.0 g), yeast extract (0.25 g), NaHCO₃(0.4 g), cysteine (0.1g), K₂HPO₄ (0.045 g), KH₂PO₄ (0.045 g), NaCl (0.09 g), (NH₄)₂SO₄ (0.09g), MgSO₄.7H₂O (0.009 g), CaCl₂) (0.009 g), resazurin (0.1 mg), hemin (1mg), biotin (1 μg), cobalamin (1 μg), p-aminobenzoic acid (3 μg), folicacid (5 μg), and pyridoxamine (15 μg).

Bacterial Strains for Use in Vaccine Compositions

The inventors have identified that the bacterial strains of theinvention are useful for treating or preventing neurodegenerativedisorders. This is likely to be a result of the effect that thebacterial strains of the invention have on the host immune system.Therefore, the compositions of the invention may also be useful forpreventing neurodegenerative disorders, when administered as vaccinecompositions. In certain such embodiments, the bacterial strains of theinvention may be killed, inactivated or attenuated. In certain suchembodiments, the compositions may comprise a vaccine adjuvant. Incertain embodiments, the compositions are for administration viainjection, such as via subcutaneous injection.

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., references[50] and [51,57], etc.

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

References to a percentage sequence identity between two nucleotidesequences means that, when aligned, that percentage of nucleotides arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 of ref[58]. A preferred alignment is determined by the Smith-Waterman homologysearch algorithm using an affine gap search with a gap open penalty of12 and a gap extension penalty of 2, BLOSUM matrix of 62. TheSmith-Waterman homology search algorithm is disclosed in ref. [59].

Unless specifically stated, a process or method comprising numeroussteps may comprise additional steps at the beginning or end of themethod, or may comprise additional intervening steps. Also, steps may becombined, omitted or performed in an alternative order, if appropriate.

Various embodiments of the invention are described herein. It will beappreciated that the features specified in each embodiment may becombined with other specified features, to provide further embodiments.In particular, embodiments highlighted herein as being suitable, typicalor preferred may be combined with each other (except when they aremutually exclusive).

MODES FOR CARRYING OUT THE INVENTION Example 1—Efficacy of BacterialInocula to Act as a Neuroprotectant

Summary

Neuroblastoma cells were treated with compositions comprising bacterialstrains according to the invention. The SH-SY5Y neuroblastoma cells usedare dopamine producing and well-established as an in vitro model forstudying neurodegenerative diseases. The ability of the bacterialstrains to increase neuroproliferation was observed. The neuroblastomacells were also treated with dopaminergic neurotoxin1-methyl-4-phenylpyridinium (MPP), which induces permanent symptoms ofParkinson's disease in neuroblastoma cells. The ability of the bacterialstrains to act as a neuroprotectant against MPP was investigated.

Material and Methods

Bacterial Strain

Megasphaera massiliensis MRx0029; Parabacteroides distasonis MRX0005

Cell Line

SH-SY5Y neuroblastoma cells were purchased from ECCACC (Cat. no:94030304) and were grown in MEM (Sigma Aldrich, cat n. M2279)supplemented with Nutrient Mixture F-12 Ham (Sigma Aldrich, cat n.N4888).

Method

Once grown the SH-SY5Y neuroblastoma cells were plated on 96-well plateat 11,000 cells/well and incubated for 2 days. The cells were thentransferred to differentiation medium (which contains FBS at 1%) and 10uM retinoic acid (Sigma Aldrich, cat. n. R2625-100MG). Differentiationmedium was replaced every other day and cells were harvested at 7 day ofdifferentiation. Cells were pre-treated with or without MPP (SigmaAldrich, cat. n. D048-1G) for 8 hours. Subsequently, cells were treatedwith 10% bacterial supernatant and incubated overnight. Cell viabilitywas measured by using CCK-8 reagent (Sigma Aldrich, Cell Counting Kit—8,cat. n. 96992-3000TESTS-F) and read at 450 nm wavelength.

Results

The results of these experiments are shown in FIG. 1. Treatment ofneuroblastoma cells with MRx0029 or MRX0005 led to an increase in theproliferation of neurons. Neuroblastoma cells that were treated with MPPtogether with the bacterial strain had increased cell viability comparedto the cells treated with MPP alone (which had decreasedviability).These data show that the bacterial strain can act as aneuroprotectant. The protective effect was greater for MRX0029-treatedcells, which rescued viability more than the positive control cellstreated with Quercetin. These data show that the bacterial strains canact as a neuroprotectant

Example 2—Efficacy of Bacterial Inocula to Reduce IL-6 Secretion

Summary

Activation of proinflammatory cytokines has been associated with neurondamage in neurodegenerative disease. Lipopolysaccharide (LPS) is a knownstimulator of the proinflammatory cytokine IL-6. Human glioblastomaastrocytoma cells were treated with compositions comprising bacterialstrains according to the invention in combination with LPS to observetheir ability to modulate the levels of IL-6.

Material and Methods

Bacterial Strain

Megasphaera massiliensis MRx0029

Cell Line

MG U373 is a human glioblastoma astrocytoma derived from a malignanttumour and were purchased from Sigma-Aldrich (cat n. 08061901-1VL). MGU373 human glioblastoma astrocytoma cells were grown in MEM (SigmaAldrich, cat n. M-2279) supplemented with 10% FBS, 1% Pen Strep, 4 mML-Glut, 1×MEM Non essential Amino Acid solution and 1× Sodium Piruvate.

Method

Once grown the MG U373 cells were plated on 24-well plate at 100,000cells/well. The cells were treated with LPS (1 ug/mL) alone or with 10%of bacteria supernatant from MRx0029 for 24h. A control was alsoperformed where the cells were incubated in untreated media. Afterwardsthe cell free supernatants were collected, centrifuged at 10,000 g for 3min at 4° C. IL-6 was measured using the Human IL-6 ELISA Kit fromPeprotech (cat n.#900-K16) according to manufacturer instructions.

Results

The results of these experiments are shown in FIG. 2. Treatment ofneuroblastoma cells with LPS and the bacteria strain led to a decreasein the level of IL-6 secreted.

Example 2b—Efficacy of Bacterial Inocula to Modulate IL-8 Secretion

Summary

As neuro-inflammation plays a pivotal role in neurodegenerative diseasesand IL-8 has been shown to have neuro-positive effects, the effect ofcompositions comprising bacterial strains of the invention and LPS onthe activation of IL-8 were assessed. Human glioblastoma astrocytomacells were treated with compositions comprising bacterial strainsaccording to the invention in combination with LPS to observe theirability to modulate the levels of IL-8.

Material and Methods

Bacterial Strains

Megasphaera massiliensis MRX0029; Parabacteroides distasonis MRX0005

Cell Line

MG U373 is a human glioblastoma astrocytoma derived from a malignanttumour and were purchased from Sigma-Aldrich (cat n. 08061901-1VL). MGU373 human glioblastoma astrocytoma cells were grown in MEM (SigmaAldrich, cat n. M-2279) supplemented with 10% FBS, 1% Pen Strep, 4 mML-Glut, 1×MEM Non essential Amino Acid solution and 1× Sodium Piruvate.

Method

Once grown the MG U373 cells were plated on 24-well plate at 100,000cells/well. The cells were treated with LPS (1 ug/mL) alone or with 10%of bacteria supernatant from MRX0029 for 24h. Afterwards the cell freesupernatants were collected, centrifuged at 10,000 g for 3 min at 4° C.IL-8 was measured using Human IL-8 ELISA Kit from Peprotech (catn.#900-K18) according to manufacturer instruction.

Results

The results of these experiments are shown in FIG. 3. Treatment ofneuroblastoma cells with the bacteria strains lead to an increase inIL-8 secretion independently of the presence of LPS.

Example 2C—Efficacy of Bacterial Inocula to Reduce α-Synuclein-InducedInflammation

Summary

Neuroinflammation plays a pivotal role in Parkinson's disease andα-synuclein has been shown to induce neuroinflammation in vivo.Therefore, the ability of the bacteria strains of the invention toinhibit α-synuclein-induced neuroinflammation was assessed. A co-cultureof human glioblastoma astrocytoma cells and neuroblastoma cells wereexposed to wild-type α-synuclein and the mutant isoforms E46K and A53Tand treated with compositions comprising bacterial strains according tothe invention. The ability of the bacteria strains to inhibitα-synuclein-induced secretion of IL-6 was then tested.

Material and Methods

Bacterial Strains

Megasphaera massiliensis MRX0029; Parabacteroides distasonis MRX0005

Cell Line

MG U373 is a human glioblastoma astrocytoma derived from a malignanttumour and were purchased from Sigma-Aldrich (cat n. 08061901-1VL). MGU373 human glioblastoma astrocytoma cells were grown in MEM (SigmaAldrich, cat n. M-2279) supplemented with 10% FBS, 1% Pen Strep, 4 mML-Glut, 1×MEM Non-essential Amino Acid solution and 1× Sodium Piruvate.

SH-SY5Y is a human neurobastoma cell line derived from a malignantneuroblastoma and can be purchased from Sigma-Aldrich (cat n.94030304-1VL). The cells were grown in 50% MEM and 50% Nutrient MixtureF-12 Ham media supplemented with 2 mM L-Glutamine, 10% heat inactivatedFBS, 100 U/ml penicillin, 100 mg/ml streptomycin. Cells on growth mediumwere plated on 96-well plate at 11,000 cells/well and placed in theincubator. After 2 days, media were replaced with differentiation medium(growth medium containing 1% FBS) and 10 μM retinoic acid.Differentiation medium was replaced every other day and cells were usedafter 7 days of differentiation.

Method

SHSY5Y cells were plated on 12 well plates at a density of 50,000cells/well. The cells were grown in 50% MEM and 50% Nutrient MixtureF-12 Ham media supplemented with 2 mM L-Glutamine, 10% heat inactivatedFBS, 100 U/ml penicillin, 100 μg/ml streptomycin. Cells on growth mediumwere plated on 96-well plate at 11,000 cells/well and placed in theincubator. After 2 days, media were replaced with differentiation medium(growth medium containing 1% FBS) and 10 μM retinoic acid.Differentiation medium was replaced every other day and cells were usedafter 7 days of differentiation. U373 were plated on 12 transwell plates(0.4 μm polyester membrane, Costar) at a density of 50,000 cells/wellfor 72 hrs. Cells were co-cultured together for 24 hrs before treatmentin differentiation medium (growth medium containing 1% FBS withoutretinoic acid).

Thereafter cells were treated with 25 μg/ml α-synuclein (Wt, A53T, E46K)in the presence or absence of 10% bacteria supernatant for 48 hrs. Cellfree Supernatants were collected, spun-down at 10000 g for 3 min at 4°C., aliquoted and stored at −80° C. Human IL-6 and IL-8 were measured asdescribed above.

Results

The results of these experiments are shown in FIG. 4. Treatment of cellswith wild-type α-synuclein and the mutant isoforms E46K and A53T inducedmoderate secretion of IL-6 (FIG. 4A). The α-syn-induced secretion ofIL-6 was inhibited in cells treated with the bacteria strains (FIG. 4A).The reduction in IL-6 secretion was greatest on administration ofMRX0029.

Example 3—Efficacy of Bacterial Inocula to Reduce NFκB Activation

Summary

Activation of the NFκB promoter leads to the production ofproinflammatory cytokines including IL-1β, IL-1α, IL-18, TNFα and IL-6.The NFκB promoter can be activated by α-synuclein and LPS by stimulatingthe TLR4 ligand. Mutations in α-synuclein, such as α-synuclein A53T, areimplicated in familial Parkinson's. Treatment of neuronal cells with LPSsimulates Parkinson's caused by environmental factors. The ability ofcompositions comprising bacterial strains according to the invention toinhibit the activation of the NFκB promoter was investigated.

Material and Methods

Bacterial Strain

Megasphaera massiliensis MRx0029

Cell Line

Human Hek blue TLR4 were purchased from InvivoGen (cat n. hkb-ht1r4).Human Hek blue TLR4 were grown in DMEM high glucose (Sigma Aldrich, catn. D-6171) supplemented with 10% FBS, 1% Pen Strep, 4 mM L-Glut,Normocin and 1×HEK Blue selection solution.

Method

Once grown the Human Hek blue cells were plated in 96 well plates at25,000 cells/well in 4 replicates. One set of cells were treated withα-synuclein A53T (1 ug/mL) alone or with 10% of bacteria supernatantfrom MRx0029 for 22h. The second set of cells were treated with LPS (10ng/mL, from Salmonella enterica serotype Typhimurium, Sigma Aldrich, catn. L6143) alone or with 10% of bacteria supernatant from MR029 for 22h.The cells were subsequently spun down and 20 ul of the supernatant wasmixed with 200ul of Quanti Blue reagent (InvivoGen, cat n. rep-qb2),incubated for 2 h and absorbance read at 655 nm.

Results

The results of these experiments are shown in FIGS. 5 and 6. FIG. 5shows that the activation of the NFκB promoter by α-synuclein isinhibited by MRx0029. FIG. 6 shows that the activation of the NFκBpromoter by LPS is inhibited by MRx0029.

Example 4—Efficacy of Bacterial Inocula to Alter Antioxidant Capacity

Summary

The ability of compositions comprising bacterial strains according tothe invention to alter the antioxidant capacity. The antioxidantcapacity of the bacterial strain was established using the well-knownABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) assay.

Bacterial Strain

Megasphaera massiliensis MRx0029

Method

Bacterial cells (10⁶ or greater) were collected and centrifuged. Theywere resuspended in assay buffer (using three times the pellet volume).The suspension was sonicated on ice for 5 minutes and then spun down at12,000×g for 10 minutes. The supernatant was removed and measured usingthe ABTS assay kit produced by Sigma Aldrich (code CS0790), inaccordance with manufacturer's instructions.

Results

The results of these experiments are shown in FIG. 7. FIG. 7 shows thatMRx0029 has an antioxidant capacity of approximately 2 mM compared toTrolox.

Example 5—Efficacy of Bacterial Inocula to Alter Lipid PeroxidationLevels

Summary

The ability of compositions comprising bacterial strains according tothe invention to alter lipid peroxidation levels was investigated. Thethiobarbituric reactive substances assay (TBARs) was used to measure theby-products of lipid peroxidation.

Material and Methods

Bacterial Strain

Megasphaera massiliensis MRx0029

Method

Bacterial cells (10⁶ or greater) were collected and centrifuged, a washstep was performed with isotonic saline before the pellet wasre-suspended in potassium chloride assay buffer. The suspension wassonicated on ice for 10 minutes and then spun down at 10,000×g for 10minutes. The supernatant was removed and the level of lipid peroxidationevaluated using the thiobarbituric reactive substances assay.

Results

The results of the experiments are shown in FIG. 8. FIG. 8 shows thatMRx029 is able to inhibit lipid peroxidation by approximately 20%, whichis a higher antioxidant capacity than the positive control, butylatedhydroxytoluene (1% w/v).

Example 6—Efficacy of Bacterial Inocula on Histone Deacetylase Activity

Summary

The ability of compositions comprising bacterial strains according tothe invention to alter histone deacetylase activity was investigated.Dysregulation of histone deacetylase has been implicated in thepathogenesis associated with age-associated neurodegenerative diseases.

Material and Methods

Bacterial Strain

Megasphaera massiliensis MRx0029

Cell Line

The cell line HT-29 was used because histone deacetylase is present.

Method

Cell free supernatants of stationary phase bacterial cultures wereisolated by centrifugation and filtering in a 0.22 uM filter. HT-29cells were used 3 days' post confluence and stepped down in 1 mL DTS 24hours prior to commencement of the experiment. The HT-29 cells werechallenged with 10% cell free supernatant diluted in DTS and was is leftto incubate for 48 hours. Nuclease proteins were then extracted usingthe Sigma Aldrich Nuclease extraction kit and samples were snap frozenprior to HDAC activity measurement. HDAC activity was assessedfluorometrically using the Sigma Aldrich (UK) kit.

Results

The results of the experiments are shown in FIG. 9. FIG. 9 shows thatMRx0029 is able reduce the levels of histone deacetylase activity.

Example 7—Level of Indole Production in Bacteria

Summary

The ability of the bacteria of the invention to produce indole wasinvestigated. Indole has been implicated in attenuating inflammation andoxidative stress.

Material and Methods

Bacterial Strain Megasphaera massiliensis MRx0029

ATCC 11775 is a bacterial reference strain that is known to produceindole.

Method

Intact bacterial cells in stationary phase were incubated with 6 mMTryptophan for 48 hours. Bacterial species which possess the enzymetryptophanase will utilise tryptophan as a substrate to produce indole.Following the 48 hour incubation period, the supernatant was removed andadded to Kovac's reagent for quantification of indole. Standards, stocksolutions and reagents were prepared using standardised methodsvalidated in-house.

Results

The results of the experiments are shown in FIG. 10. FIG. 10 shows thatMRx0029 has the capacity to produce indole from tryptophan, atconcentrations of approximately 0.2 mM.

Example 8—Level of Kynurenine Production in Bacteria

Summary

The ability of the bacteria of the invention to produce kynurenine wasinvestigated. Dysregulation of the kynurenine pathway can lead toactivation of the immune system and the accumulation of potentiallyneurotoxic compounds. Alterations in the kynurenine metabolism may beinvolved in the development of Parkinson's diseases.

Bacterial Strain

Megasphaera massiliensis MRx0029

DSM 17136 is a strain of Bacteroides copricola that is known to producekynurenine.

Method

Cell free supernatants of stationary phase bacterial cultures wereisolated by centrifugation and filtering in a 0.22 uM filter and frozenuntil use. Kynurenine standards, stock solutions and reagents wereprepared using standardised methods validated in-house. Sample weretreated with trichloroacetic acid and centrifuged at 10,000×g for 10minutes at 4° C. The supernatant was collected and dispensed into a 96well plate. Ehrlich's reagent was used for kynurenine detection andadded at a ratio of 1:1.

Results

The results of the experiments are shown in FIG. 11. FIG. 11 shows thatMRx0029 has the capacity to produce kynurenine at a concentration ofapproximately 40 μM.

Example 9—Levels of Dopamine, DOPAC and HVA in Striatum inBacteria-Treated MPTP Mice

Parkinson's disease is a common neurodegenerative disorder whosecardinal clinical features include tremor, slowness of movement,stiffness, and postural instability. These symptoms are primarilyattributable to the degeneration of dopaminergic neurons in thesubstantia nigra pars compacta and the consequent loss of theirprojecting nerve fibers in the striatum [60]. Mice treated with MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) selectively losesignificant numbers of nigrostriatal dopaminergic neurons [61]. MPTPinduced loss of dopaminergic cells in substantia nigra mimics theclinical condition in Parkinson's disease and is therefore a usefulmodel to test anti-parkinsonian drugs.

The aim of this study was to evaluate the effects of MRX0029 anaerobicbacteria using MPTP lesioned mice.

48 male mice were allocated to 4 different treatment groups (groups A,B, E and I, with n=12 animals in each group). The treatment groups areshown in Table 1 below and the project time course is outlined below.

TABLE 1 Treatment groups Treatment Lesion Group n Substance Safety levelDose Route Schedule Substance Dose Route Schedule A 12 Vehicle (PBS) — —p.o. 18 days: day Vehicle (0.9% i.p. day 0 (−14)-day 3 saline) B 12Vehicle (PBS) — — p.o. 18 days: day MPTP 4 × 20 i.p. day 0 (−14)-day 3mg/kg E 12 MRx0029 S1/S2 2 × 10{circumflex over ( )}8 p.o. 18 days: dayMPTP 4 × 20 i.p. day 0 Megasphaera bacteria (−14)-day 3 mg/kg sp. (gly)I 12 Vehicle (PBS) — — p.o. 18 days: day MPTP 4 × 20 i.p. day 0(−14)-day 3 mg/kg 7-nitroindazole — 50 mg/kg i.p. day 0 (2 × i.p.)

Groups A, B, E and I were treated daily for 18 days via oral gavage witheither bacteria (MRx0029—group E), or vehicle (PBS). Oral treatmentstarted 14 days before MPTP lesion. Group I animals received a dailyvehicle (PBS) p.o. (per oral) treatment and were injected i.p.(intraperitoneal) with the reference drug 30 min before and 90 min afterfirst MPTP on day 0. The application volume for p.o. and vehicletreatment was 200 μl per mouse. Bacteria strain of group E was fromglycerol stocks (gly). For oral treatment, gavages for applications werestored in vial containing 70% Ethanol and were flushed before and aftereach use with distilled water. Every treatment group had its own gavageand ethanol vial and distilled water vial. The tubes and gavages werenot changed between the groups. Directly before treatment each syringewas flushed with N2.

On day 0 MPTP (20 mg/kg bodyweight (b.w.) 4 times, 2h inter-treatmentinterval) was injected i.p. in animals of groups B, E and I. One groupof animals (A) was sham lesioned by i.p. administration of the MPTPvehicle (0.9% saline). The application volume was 10 μl per g bodyweight. Weighing of the animals was performed before the MPTP treatmentto dose the animals according to their actual body weight. Afterwardsanimals received the daily p.o. treatment.

Formulation of Preparations for Dosing and Preparation of GlycerolStocks for Dosing

Name of the Bacteria strain: MRx0029 Megasphaera sp. Storagecondition/stability: -80° C. Vehicle: 1× PBS Treatment dosages: 2 ×10{circumflex over ( )}8 bacteria Administration: 200 μl Lot Number: n/a

For Treatment Group E (MRx0029)

1.) 1 glycerol stock was taken from the −80° C. freezer and placed underanaerobic conditions (anaerobic jar with sachet) at 37° C. in order tothaw (this took 30-40 mins).

2.) The completely thawed glycerol stock was centrifuged at 6000×g for10 min at room temperature.

3.) The supernatant was discarded without disturbing the pellet (e.g.using a pipette).

4.) 4.22 mL of sterile pre-warmed (37° C.) 1×PBS was added and gentlymixed using a pipette.

5.) The mice were dosed with 200 μL of the bacterial solution. Theanimals were dosed within 15 mins after resuspension of the pellet withPBS.

Reference Drug Group Formulation

Name of the Reference item: 7-Nitroindazole Storage condition/stability:-20° C. Vehicle: Peanut oil Treatment dosages: 50 mg/kg Administration:i.p. (30 min before and 90 min after 1^(st) MPTP treatment) BatchNumber: MKBS6671V

The appropriate amount of 7-Nitroindazole was dissolved in peanut oil toreach the final concentration of 50 mg/kg.

Materials and Methods

Animals

Mouse line: C57BL/6J (JAX ™ Mice Strain) Provider: Charles RiverLaboratories Age at start: ~10 weeks Sex: Male Number of animals: 48

Specific Handling of Animals and Randomization

Gloves were changed between each treatment group and sprayed with 70%ethanol solution between each cage of the same group to minimize therisk of contamination whenever animals were handled (e.g.: treatment,behavioural testing, cleaning and tissue sampling).

The treatment was at random and alternated daily so as to prevent thesame groups being treated at the same time each day. Animals wererandomized per cage at the tissue sampling.

Tissue Sampling and Processing

On day 4 animals of all groups were sacrificed and brains werecollected. Therefore, mice were deeply anesthetized by Pentobarbitalinjection (600 mg/kg).

Blood (approximately 500 μl) was collected by heart puncture. Mice werethen transcardially perfused with 0.9% saline and brains were removedand hemisected. The left hemisphere was subdivided into striatal tissue(for HPLC), substantia nigra tissue as well as residual brain, weighedand immediately frozen and stored at −80° C. Instruments and surfaceswhich were in contact with the animals had to be cleaned with 70%ethanol before the next animal was dissected.

Biochemical Analysis of Dopamine, DOPAC and HVA Levels with HPLC inStriatum

The striatal samples (n=6 from each treatment group; total 24 samples)were mixed at a ratio of 1:10 (w/v) with 0.2 M perchloric acid including100 μM EDTA-2Na and homogenized at 0° C. in aglass-pestlemicro-homogenizer. Following standing for 30 min on ice, thehomogenates were centrifuged at 10,000 RPM for 10 minutes in arefrigerated centrifuge Biofuge Fresco (Heraeus Instruments, Germany).The supernatants were carefully aspirated and mixed with 0.4 MNa-acetate buffer, pH 3 at a ratio 1:2 (v/v) and filtered through a 0.22μm centrifugal filter (Merck Millipore, Germany) for 4 min at 14 000 gat 4° C. The filtrates were stored at −80° C. before HPLC analysis.

HPLC Analysis

Concentrations of DA, DOPAC and HVA in the striatal samples weredetermined by column liquid chromatography with electrochemicaldetection [62; 63]. The HPLC system (HTEC-500, Eicom Corp., Kyoto,Japan) including a pulse-free microflow pump, a degasser and anamperometric detector equipped with a glassy-carbon electrode operatingat +0.45 V vs. an Ag/AgCl ref. electrode was used. Samples were injectedby use of a CMA/200 Refrigerated Microsampler (CMA/Microdialysis,Stockholm, Sweden). The chromatograms were recorded and integrated byuse of a computerized data acquisition system (DataApex, Prague, CzechRepublic). DA, DOPAC and HVA were separated on a 150×2.1 i.d. mm column(CA5-ODS, Eicom Corp., Kyoto, Japan). The mobile phase consisted of 0.1M phosphate buffer at pH 6.0, 0.13 mM EDTA, 2.3 mMsodium-1-octanesulfonate and 20% (v/v) methanol. The detection limit(signal-to-noise ratio=3) for DA was estimated to 0.5 fmol in 15 μl(0.03 nM) injected onto the column.

Results

Administration of bacteria strains was well tolerated by the animals. Onthe MPTP lesion day and if necessary on the day afterwards a red lightwas used to warm the animals. If animals were in bad conditions (feltcold, dehydrated, abnormal behaviour), they were supplied with wet foodand subcutaneous saline treatment if necessary.

For analysis of Dopamine, DOPAC and HVA levels, striatal tissue of 6animals per treatment group were used. Data were analyzed by usingKruskal-Wallis test followed by Dunn's multiple comparison post hoc testor One-way analysis of variance followed by Bonferroni post hoc test (Avs. all(*), B vs. all, I vs. all (#)). */#=p<0.05; **=p<0.01;***=p<0.001.

The healthy animals in group A had high levels of Dopamine, DOPAC andHVA whereas MPTP treatment in group B reduced this and the positivecontrol (group I) recovered the production to some degree (FIG. 12).Animals of group I tended to have higher Dopamine levels than thebacteria treated group and group B. DOPAC (a Dopamine metabolite) levelsin general were significantly lower in animals of group B compared toDOPAC levels of unlesioned animals of group A (FIG. 12B).

Significantly, treatment with MRx0029 (group E) was found to recoverproduction of Dopamine and DOPAC (FIGS. 12A and 12B, respectively).Treatment with MRx0029 may therefore be useful for treating orpreventing neurodegenerative disorders.

Example 10—Efficacy of Bacteria to Alter Neurite Outgrowth

Summary

Neurite outgrowth is an important process for the development ofconnections between neurons. The ability of bacterial strains andorganic acids to induce neurite outgrowth was therefore tested bymeasuring transcriptional levels of microtubule associated protein MAP2,a specific neuronal differentiation marker.

Bacterial Strain

Megasphaera massiliensis MRX0029.

Method

SHSY5Y were plated in 10 cm petri dishes a density of 2×10⁶ cells. After24h cells were treated in differentiation medium (growth mediumcontaining 1% FBS without RA) with 10% bacteria supernatants or YCFA+,10 uM RA, 200 uM hexanoic acid or 200 uM valproic acid, for 17 hrs.There after representative images were taken using phase contrast EVOSXL core microscope at 40×/0.65 magnification. Cells were collected, andtotal RNA was isolated according to RNeasy mini kit protocol (Qiagen).cDNAs were made using the high capacity cDNA reverse transcription kit(Applied Biosystems). Gene expression was measured using qPCR. GAPDH wasused as internal control. Fold change was calculated according to the2^((−ΔΔct)) method.

Immunofluorescence and Confocal Microscopy

Cells were seeded onto 8 well chamber slides (Marienfeld LaboratoryGlassware) at 5×10⁴ cells/well overnight and were treated with 10%bacterial supernatant for 24 hrs. For differentiation, cells weretreated with 10 nM Retinoic acid for 5 days before treating withbacterial supernatant. Cells were then fixed with 4% paraformaldehyde inPBS for 20 minutes at room temperature (RT). Fixed cells were washedwith PBS, and permeabilized with 1% Triton X-100 in PBS for 10 minutes.After washing with PBS, the slides were incubated with blocking buffer(4% BSA/PBS) for 1 hr at RT before adding anti-MAP2 antibody (sc-74421,Santa Cruz Biotechnology Inc) diluted in 1% BSA/PBS for 12 hr at 4° C.They were then washed twice with PBS, followed by incubation with AlexaFlour 488 conjugated anti-mouse (Molecular Probes Inc) and Alexa Flour594 conjugated Phalloidin (ab176757, Abcam) for 1 hr at RT. Afterwashing 3× with PBS, the slides were mounted with Vectorshield□containing DAPI (Sigma, Aldrich). Slides were viewed using a ZeissAxioscope microscope equipped with a 63×/1.2 W Korr objective and filtersets suitable for detection of the fluorochromes used. Manual exposuretimes for the digital acquisition of images immuno-labelled with MAP-2were kept constant allowing comparison between different wells andtreatments. Phalloidin (F-actin) and DAPI exposure times varied to suitthe field of view. Randomised fields of view were acquired using aQImaging camera controlled by Image Pro Plus software. Images were savedas TIFs and opened in Adobe Photoshop CC 2015.1.2 and overlays of theMAP-2, DAPI and Phalloidion images overlaid and merged. Representativeimages were selected to illustrate the differences in abundance andlocation of the proteins examined

Results

The results are shown in FIG. 13. FIG. 13A shows representativemicroscopy images of undifferentiated SHSY-5Y cells incubated with eachof the acids and bacteria supernatants. Treatment of cells with MRX0029induced a neuron-like phenotype, showing similar features to cellstreated with retinoic acid (which is used for terminal differentiationof neuroblastoma cells), where cell bodies are bigger andpyramidal-shaped, with neurites and processed branching out to networkwith neighbour cells. FIG. 13B shows that MRx0029 significantlyupregulates MAP2 in undifferentiated neuroblastoma cells. Phalloidin (anactin cytoskeleton-binding agent) staining further proved a differentarrangement of cytoskeletal structure in cells treated with MRx0029,further supporting the neuronal differentiation hypothesis for MRx0029(FIG. 13B).

Example 11—Efficacy of Bacterial Inocula to Reduce Oxidative Levels inCells

Background

The generation of reactive oxygen species contributes to the pathologyof neurodegenerative diseases. The ability of bacterial strains toprotect differentiated SHSY-5Y and U373 cells from reactive oxygenspecies (ROS) generated by treatment with Tert-Butyl Hydrogen Peroxide(TBHP) was investigated.

Material and Methods

Bacterial Strain

Megasphaera massiliensis MRX0029

Method

SHSY-5Y cells were plated in black flat bottom 96 well plate at densityof 5000 cells/well and placed in the CO2 incubator. After 24 h, mediawere replaced with differentiation medium (growth medium containing 1%FBS) and 10 μM retinoic acid. Differentiation medium was replaced everyother day. On Day 10 the differentiation medium was removed and cellswere washed with pre-warmed PBS and stained with 10 uM DCFDA molecularprobe for 20 mins in growth medium containing 1% FBS. Then cells werewashed with pre-warmed PBS again and treated with 100 uM TBHP in thepresence or absence of 10% bacteria supernatant for 2h. Fluorescenceintensity was measured using TECAN plate reader at Ex/Em 485/530 nm.

Results

The results of the experiments are shown in FIG. 14. FIG. 14b shows thatMRX0029 is able to inhibit ROS production in differentiated SHSY-5Yneuroblastoma cells. MRX0029 did not have an effect on the generation ofROS in U373 astroglioblastoma cells (FIG. 14a ). This shows that thisaspect of the antioxidant effect is neuron-specific.

Example 12—Neuroprotection

RA-differentiated SHSY-5Y cells were treated with MPP+, the activemetabolite of MPTP, a chemical widely used to mimic in vitro and in vivosome of the features of PD pathology. Cell viability was measured as therate of mitochondria respiration (FIG. 15). Both MRx0005 and MRx0029showed significant effects and promote per se an increase of themitochondria metabolic activity in SHSY-5Y cells. MRX0029 showedcomplete protection from MPP+, restoring cell viability nearly to thesame level of untreated cells and higher than quercetin positivecontrol. MRx0005 protection was about 20% compared to YCFA-MPP+ treatedsample, about the same observed for the quercetin positive control (FIG.15).

Example 13—Further Analysis of the Mechanism of Histone DeacetylationInhibition

Introduction

The gut microbiota, with its immense diversity and metabolic capacity,represents a huge metabolic reservoir for production of a vast varietyof molecules with potential to influence HDAC activity. Few studies haveassessed the HDAC inhibitory activity of microbially-derived metabolitesother than butyrate, which has been shown to inhibit HDAC and isassociated with improvement of motor function in Huntington's disease[64]. The inventors therefore sought to determine which metabolites areresponsible for HDAC inhibition and further elucidate the mechanisms bywhich inhibition is achieved.

Material and Methods

Bacterial Culture and Cell-Free Supernatant Collection

Pure cultures of bacteria were grown anaerobically in YCFA broth untilthey reached their stationary growth phase. Cultures were centrifuged at5,000×g for 5 minutes and the cell-free supernatant (CFS) was filteredusing a 0.2 μM filter (Millipore, UK). 1 mL aliquots of the CFS werestored at −80° C. until use. Sodium butyrate, hexanoic and valeric acidwere obtained from Sigma Aldrich (UK) and suspensions were prepared inYCFA broth.

SCFA and MCFA Quantification of Bacterial Supernatants

Short chain fatty acids (SCFAs) and medium chain fatty acids (MCFAs)from bacterial supernatants were analysed and quantified by MS Omics APSas follows. Samples were acidified using hydrochloride acid, anddeuterium labelled internal standards where added. All samples wereanalyzed in a randomized order. Analysis was performed using a highpolarity column (Zebron™ ZB-FFAP, GC Cap. Column 30 m×0.25 mm×0.25 μm)installed in a GC (7890B, Agilent) coupled with a quadropole detector(59977B, Agilent). The system was controlled by ChemStation (Agilent).Raw data was converted to netCDF format using Chemstation (Agilent),before the data was imported and processed in Matlab R2014b (Mathworks,Inc.) using the PARADISe software described in [65].

Specific HDAC Activity Analysis

Specific HDAC inhibition activity was analysed for HDAC1, 2, 3, 4, 5, 6,9 using fluorogenic assay kits for each type of HDAC (BPS Bioscience,CA). Assays were conducted according to manufacturer's instructions andeach sample were performed in replicates. Cell free supernatants werediluted 1 in 10 and exposed to specific HDAC proteins provided in thekit to maintain consistency between methods.

Results

Histone Deacetylase-Inhibiting Gut Commensal Microbial Metabolites areButyrate and Valeric Acid

MRx0029, whose supernatant showed strong HDAC inhibition in both HT29whole cells and HT29 cell lysates, produced valeric acid and hexanoicacid at mean concentrations of 5.08 mM and 1.60 mM, respectively (FIGS.16A and C).

To investigate which metabolites were responsible for the strain-inducedHDAC inhibition, different concentrations of hexanoic acid, valeric acidand sodium butyrate were measured for their HDAC inhibition on wholeHT-29 cells and on HT-29 cell lysate. The results in FIG. 16B showsignificant (P<0.05) inhibition of HDAC activity by sodium butyrate onwhole cells as well as on the cell lysate, while hexanoic acid did notshow significant inhibitory activity. Valeric acid inhibited total HDACactivity (* (p<0.05), ** (p<0.005), *** (P<0.001), **** (p<0.0001)).

Potent Total HDAC Inhibitors Investigated Target Class I HDACs.

The specific HDAC inhibition profile of the test bacteria strain wasinvestigated. Specific HDAC inhibition assays (BPS Bioscience, CA) werecarried out for Class I and Class II HDACs. The ability of the bacterialstrain to inhibit HDAC enzymes was compared to butyrate, hexanoic andvaleric acid. Our results demonstrate that MRX0029, is a very potentinhibitor of Class 1 HDAC enzymes (HDAC1, 2 and 3). Inhibition of classII HDACs was not as significant (data not shown).

Discussion

The strain with HDAC inhibitory activity produced significant amounts ofvaleric acid and hexanoic acid as well as significant amounts of sodiumbutyrate (FIG. 16C). When tested as pure substances, valeric acid andsodium butyrate resulted in significant HDAC inhibition (p<0.0001).

Interestingly, the results for specific HDAC activity show that thetested strain is a potent inhibitor of Class I HDACs, and particularlyHDAC2 (FIGS. 17 and 18). Class I HDACs (HDAC1, 2, 3 and 8) reside in thenucleus and are ubiquitously expressed in several human cell types.HDACs 1-3 share more than 50% homology, but have distinct structures andcellular functions [66]. They are primarily involved in cell survival,proliferation and differentiation, and thus their inhibition may beuseful is wide array of diseases [67]; [68]; [69]; [70]; [71].

Example 14—Level of BDNF Secretion in SHSY-5Y Cells

Background

Brain-derived neurotrophic factor (BDNF) is a ubiquitous molecule in thebrain associated with neural development, neuro-protection andneuro-regeneration. BDNF not only protects against neurodegeneration butalso mental disorders like depression and anxiety, which are quitecommon amongst patients diagnosed with PD or AD.

Methods

SH-SY5-SY were plated in 24 wells plate at density of 60,000 cells/welland placed in the incubator. After 24 h, media were replaced withdifferentiation medium (growth medium containing 1% FBS) and 10 μMretinoic acid. Differentiation medium was replaced every other day andcells were used on day 10 of differentiation. For the treatmentdifferentiation medium was removed and replaced with 450u1 of fullgrowth media and 50 μl of bacteria SN was added to the treated wells orYCFA+ was added as negative Control.

Results

The results are shown in FIG. 19, which shows that administration ofMRX0029 in combination with retinoic acid increases the secretion ofBDNF from differentiated neuroblasoma cells. Compositions comprisingcommensal bacteria and organic acids may therefore be useful in therapy.

Example 15—Metabolite Production—Metabolites in the Brain BACKGROUND

Metabolites present in bacteria supernatants can directly influence thehost response to oxidative stress, cell-to-cell communication andneuroprotection. Metabolites that play a key role in neurologicalprocesses were measured during the ex vivo screening in brain tissue ofmice fed with MRx0005 and MRx0029.

Methods

Animals

BALBc (Envigo, UK) adult male mice were group housed under a 12 hlight-dark cycle; standard rodent chow and water were available adlibitum. All experiments were performed in accordance with Europeanguidelines following approval by University College Cork Animal EthicsExperimentation Committee. Animals were 8 weeks old at the start of theexperiment.

Study Design

Animals were allowed to habituate to their holding room for one weekafter arrival into the animal unit. They receive oral gavage (2000 μLdose) of live biotherapeutics at a dose of 1×10⁹ CFU for 6 consecutivedays between 15:00 and 17:00. On day 7, the animals are decapitated, andtissues are harvested for experimentation.

Tissue Collection

Animals were sacrificed in a random fashion regarding treatment andtesting condition; sampling occurred between 9.00 a.m. and 1:00 p.m.Trunk blood was collected in potassium EDTA (Ethylene Diamine TetraAcetic Acid) tubes and spun for 15 min at 4000 g. Plasma was isolatedand stored at −80° C. for further analysis. The brain was quicklyexcised, dissected and each brain region was snap-frozen on dry ice andstored at −80° C. for further analysis. Spleen was removed and processedimmediately after culls for ex-vivo immune stimulation. Intestinaltissue (2 cm segments of ileum and colon closest to the caecum wereexcised, and the furthest 1 cm of tissue from the caecum were used) weremounted into the Using chambers for intestinal permeability assay. Thecaecum was removed, weighted and stored at −80° C. for SCFAs analysis.

Monoamine Analysis

Neurotransmitter concentration was analysed by HPLC on samples from thebrainstem. Briefly, brainstem tissue was sonicated in 500 μl of chilledmobile phase spiked with 4 ng/40 μl of N-Methyl 5-HT (Sigma ChemicalCo., UK) as internal standard. The mobile phase contained 0.1 M citricacid, 5.6 mM octane-1-sulphonic acid (Sigma), 0.1 M sodium dihydrogenphosphate, 0.01 mM EDTA (Alkem/Reagecon, Cork) and 9% (v/v) methanol(Alkem/Reagecon) and was adjusted to pH 2.8 using 4 N sodium hydroxide(Alkem/Reagecon). Homogenates were then centrifuged for 15 min at22,000×g at 4° C. and 40 μl of the supernatant injected onto the HPLCsystem which consisted of a SCL 10-Avp system controller, LECD 6Aelectrochemical detector (Shimadzu), a LC-10AS pump, a CTO-10A oven, aSIL-10A autoinjector (with sample cooler maintained at 40 C) and anonline Gastorr Degasser (ISS, UK). A reverse-phase column (Kinetex 2.6 uC18 100×4.6 mm, Phenomenex) maintained at 30° C. was employed in theseparation (Flow rate 0.9 ml/min). The glassy carbon working electrodecombined with an Ag/AgCl reference electrode (Shimdazu) operated a +0.8V and the chromatograms generated were analyzed using Class-VP 5software (Shimadzu). The neurotransmitters were identified by theircharacteristic retention times as determined by standard injections,which run at regular intervals during the sample analysis. The ratios ofpeak heights of analyte versus internal standard were measured andcompared with standard injection. Results were expressed as ng ofneurotransmitter per g fresh weight of tissue.

Metabolite Analysis

For GC-metabolite analysis, samples of bacterial supernatants werederivatized with methyl chloroformate using a slightly modified versionof the protocol described in [72]. All samples were analyzed in arandomized order. Analysis was performed using GC (7890B, Agilent)coupled with a quadropole detector (59977B, Agilent). The system wascontrolled by ChemStation (Agilent). Raw data was converted to netCDFformat using Chemstation (Agilent), before the data was imported andprocessed in Matlab R2014b (Mathworks, Inc.) using the PARADISe softwaredescribed in [65].

For fatty acid analysis samples were acidified using hydrochloride acid,and deuterium labelled internal standards where added. All samples wereanalyzed in a randomized order. Analysis was performed using a highpolarity column (Zebron™ ZB-FFAP, GC Cap. Column 30 m×0.25 mm x 0.25 μm)installed in a GC (7890B, Agilent) coupled with a quadropole detector(59977B, Agilent). The system was controlled by ChemStation (Agilent).Raw data was converted to netCDF format using Chemstation (Agilent),before the data was imported and processed in Matlab R2014b (Mathworks,Inc.) using the PARADISe software described in [65].

Results—Neurotransmitter Production

The results are shown in FIG. 20, which shows that in brains of mice fedwith MRx0029, noradrenaline levels are increased (p=0.0507), accompaniedwith a slight increase of serotonin and 5-HIAA. These data support themetabolite analysis set out below, suggesting that MRx00029 is a majorproducer of 4-hydroxyphenylacetic acid, a known antioxidant [73]. Moreimportantly, 4-hydroxyphenylacetic acid is a synthetic intermediate ofdopamine and norepinephrine and an important bio-active molecule [74].In fact, in PD, degenerative changes extend beyond the dopaminergicsystem, affecting equally the serotonergic and noradrenergic systems,which in turn leads to decreased levels of serotonin(5-hydroxytryptamine, 5-HT) and noradrenaline (norepinephrine) in bothstriatal and extra-striatal structures [75]. L-DOPA targets mainly thedopamine-related features of PD, however it does not address thedecreases in both 5-HT and noradrenaline. Adding to this is that thelonger is the duration of L-DOPA treatment, the more visible are a rangeof motor and nonmotor complications (e.g. dyskinesia, psychiatricsymptoms) [76]. Therefore, these data demonstrate that bacteria thatproduce organic acids, such as 4-hydroxyphenylacetic acid, may be usefulin therapy, in particular in the treatment of neurodegenerativediseases.

Results—Metabolite Production

Metabolites present in bacteria supernatants can directly influence thehost response to oxidative stress, cell-to-cell communication andneuroprotection in the specific. Metabolites in the supernatant ofcultures of MRX0029 and MRX0005 were analysed and the results are shownin FIG. 21.

A few metabolites showed a striking difference between the two strainsanalysed. The concentration of succinic acid was particularly elevatedin MRx0005. Interestingly, the ratio sample/media for4-hydroxyphenylacetic acid was significantly higher in MRx0029 (FIG.21A).

Fatty acid analysis in the supernatants revealed an interestingdichotomy in the two strains: MRx0005 produced mainly acetic andpropanoic acid, while MRx0029 produced butanoic, pentanoic and hexanoicacid, both in the linear and branched forms (FIG. 21B). The two strainslooked very different and in particular, the production of succinic acidand 4-hydroxyphenylacetic acid by MRx0005 and MRx0029 respectively wasnotable (FIG. 21A). Furthermore, MRx0005 seems to produce more C2 and C3short chain fatty acids, while MRx00029 produced more C4 (butyrate) andboth linear and branched medium chain fatty acids, including hexanoicacid.

Succinic acid is a Krebs cycle metabolite involved in oxidativephosphorylation. Oxidative phosphorylation complex is a key step forsynaptic trafficking of proteins and vesicles to proximal and distalregions [77]. Its dysfunction has been reported in neurodegenerativedisorders including Alzheimer's disease, Parkinson's disease andSpinocerebellar ataxia type 1 [78]. These findings are particularlyinteresting as succinic acid can augment mitochondrial activity andsupport vulnerable neurons in neurodegenerative disease related tomisfolded proteins including PD [79]. BDNF and succinic acid have both asimilar protective activity not only in neuro-degeneration but also inmental disorders like depression and anxiety, which are quite commonamongst patients diagnosed with PD or AD.

FIG. 21B also demonstrates that MRX0029 is a butyrate (butanoic acid)producer. This may be significant because butyrate has a known role isreducing impermeability of the blood brain barrier, which has aneuroprotective effect [80]. This property of MRx0029 (and otherneuroprotective bacteria) may contribute to its efficacy.

Example 16—Modulation of the mRNA Expression of Tight Junction Proteinsby MRx0029

Since recent evidence suggests that intestinal dysfunction andinflammation is a non-motor symptom associated with PD, the ability ofthe bacterial strains of the invention to cause any intestinal barrierdysfunction was investigated. HT29-mtx epithelial, mucin-producing cellmonolayers [81] were used as an in vitro model to evaluate gut barrierdisruption and immune stimulation following treatment with MRx0005 andMRx0029. Differentiated HT29-mtx cells exposed to phorbol12-myristate-13-acetate (PMA) secreted a significant amount of IL-8; incontrast treatment for 24h with MRx005 and MRx0029 bacterialsupernatants, induced an even lower secretion of IL-8 compared than bothuntreated and YCFA-treated cells (FIG. 22A).

The ability of MRx0005 and MRx0029 to regulate epithelial permeabilityby modifying intracellular signal transduction involved in theexpression and localization of proteins involved in the gut barrierformation was then investigated.

RNA was isolated and Quantitative RT-PCR (qRT-PCR) analysis wasperformed to characterize the changes in gene expression of tightjunction proteins during incubation with MRx0005 and MRx0029. Theadministration of MRx0029 enhanced Occludin, Vlillin, Tight JunctionProtein 1 and 2 (respectively TJP1 and TJP2) mRNA expression after 2hincubation (FIG. 22B). In contrast, exposure to MRx0005 did not alterthe gene expression of tight junction proteins indicating that the twostrains act differentially on the intestinal barrier.

The in vitro results were compared with data from the ex vivo parallelanalysis on the gut of mice fed with MRx0005 and MRx0029. Geneexpression of TJP2 and occludin was quantified in the colon and ileum.The ex vivo data perfectly mirror the in vitro data as MRx0029 was ableto significantly up-regulate TJP1 and Occludin (p=0.073) in the colonregion of the murine intestine (FIG. 22C+22D). MRx0029 was also able todecrease the permeability function in the colon of the same mice (FIG.22E+22F).

Materials and Methods—RNA Extraction and qPCR Analysis

Total RNA was extracted using the RNeasy mini kit (Qiagen, Manchester,JUK) according to the manufacturer's instructions, and the RNAconcentration determined by absorbance at 260/280 nm using aspectrophotometer (nano-Drop ND-1000; Thermo Scientific, Wilmington,Del.). For mRNA expression analysis, cDNA was prepared from total RNAusing the High-Capacity cDNA reverse transcription kit (AppliedBiosystems, UK) according to the manufacturer's instructions. Thereverse transcription reactions were performed in a Thermo cycler(Biometra, Germany) at 25° C. for 10 min, 37° C. for 120 min, and 85° C.for 5 min, hold on at 4° C. Resulting cDNA was amplified in duplicatesby the SYBR-Green PCR assay, and products were detected on QuantStudio 6flex real-time PCR machine (Applied Biosystems, UK) using a standardisedprofile (initial denaturation of 95° C. for 10 minutes, followed by 40cycles of 15 seconds of denaturation at 95° C. and 60 seconds ofannealing/extension at 60/65° C., depending on the primers. Adissociation stage was added after the 40 cycles to generate a meltingcurve. Analysis was performed using the Applied Biosystems QuantStudioReal-Time PCR Software v1.2. The primer sequences for Actin, Villin,Occludin TJP1 and TJP2 are provided in the sequence listing.

Example 16—Stability Testing

A composition described herein containing at least one bacterial straindescribed herein is stored in a sealed container at 25° C. or 4° C. andthe container is placed in an atmosphere having 30%, 40%, 50%, 60%, 70%,75%, 80%, 90% or 95% relative humidity. After 1 month, 2 months, 3months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years, atleast 50%, 60%, 70%, 80% or 90% of the bacterial strain shall remain asmeasured in colony forming units determined by standard protocols.

Example 17

Methods

Animals

The animals and study design used were the same as for Example 15.

Bacterial Strains

-   -   755: Parabacteroides distasonis (MRX005)    -   Megasphaera massiliensis (MRX0029)

Tissue Collection

Animals were sacrificed in a random fashion regarding treatment andtesting condition; sampling occurred between 9.00 a.m. and 2:30 μm.Trunk blood was collected in potassium EDTA (Ethylene Diamine TetraAcetic Acid) tubes and spun for 15 min at 4000 g. Plasma was isolatedand stored at −80° C. for further analysis. The brain was quicklyexcised, dissected and each brain region was snap-frozen on dry ice andstored at −80° C. for further analysis. Spleen was removed, collected in5 mL RPMI media (with L-glutamine and sodium bicarbonate, R8758Sigma+10% FBS (F7524, Sigma)+1% Pen/Strep (P4333, Sigma)) and processedimmediately after culls for ex-vivo immune stimulation. Intestinaltissue (2 3 cm segments of ileum and colon closest to the caecum wereexcised, and the furthest 1 cm 2 cm of tissue from the caecum were used)were mounted into the Ussing chambers for intestinal permeability assay.The caecum was removed, weighted and stored at −80° C. for SCFAsanalysis.

Monoamine Analysis

The neurotransmitter concentration was analysed as described in Example10

Spleen Cytokine Assay

Spleens were collected immediately in 5 mL RPMI media followingsacrifice and cultured immediately. Spleen cells were first homogenisedin this RPMI media, followed by 5 mins incubation with 1 ml of RBC lysisbuffer (11814389001 ROCHE, Sigma). A further 10 ml of RPMI media wasadded, followed by 200G centrifugation for 5 mins. The supernatant wasthen filtered through 40 um strainer. Cells were counted and seeded(4,000,000/mL media). After 2.5 h of adaptation, cells were stimulatedwith lipopolysaccharide (LPS-2 μg/ml) or concanavalin A (ConA-2.5 μg/ml)for 24 h. Following stimulation, the supernatants were harvested toassess the cytokine release using Proinflammatory Panel 1 (mouse) V-PLEXKit (Meso Scale Discovery, Maryland, USA) for TNFα, IL-10, IL-1β,Interferon γ, CXCL2 and IL6. The analyses were performed using MESOQuickPlex SQ 120, SECTOR Imager 2400, SECTOR Imager 6000, SECTOR S 600.

Gene Expression Analysis

Total RNA was extracted using the mirVana™ miRNA Isolation kit(Ambion/Llife technologies, Paisley, UK) and DNase treated (TurboDNA-free, Ambion/life technologies) according to the manufacturersrecommendations. RNA was quantified using NanoDrop™ spectrophotometer(Thermo Fisher Scientific Inc., Wilmington, Del., USA) according to themanufacturer's instructions. RNA quality was assessed using the AgilentBioanalyzer (Agilent, Stockport, UK) according to the manufacturer'sprocedure and an RNA integrity number (RIN) was calculated. RNA with RINvalue >7 was used for subsequent experiments. RNA was reversetranscribed to cDNA using the Applied Biosystems High Capacity cDNA kit(Applied Biosystems, Warrington, UK) according to manufacturer'sinstructions. Briefly, Multiscribe Reverse Transcriptase (50 U/μL)(1)(2)(1)(10) was added as part of RT master mix, incubated for 25° C.for 10 min, 37° C. for 2 h, 85° C. for 5 min and stored at 4° C.Quantitative PCR was carried out using probes (6 carboxyfluorescein-FAM) designed by Applied Biosystems to mouse specifictargeted genes, while using β-actin as an endogenous control.Amplification reactions contained 1 μl cDNA, 5 μl of the 2×PCR Mastermix (Roche), 900 nM of each primer and were brought to a total of 10 μlby the addition of RNase-free water. All reactions were performed intriplicate using 96-well plates on the LightCycler®480 System. Thermalcycling conditions were as recommended by the manufacturer (Roche) for55 cycles. To check for amplicon contamination, each run contained notemplate controls in triplicate for each probe used. Cycle threshold(Ct) values were recorded. Data was normalized using β-actin andtransformed using the 2−ΔΔCT method and presented as a fold change vs.control group.

Short Chain Fatty Acids Analysis in the Caecal Content

Caecum content was mixed and vortexed with MilliQ water and incubated atroom temperature for 10 min. Supernatants were obtained bycentrifugation (10000 g, 5 min, 4° C.) to pellet bacteria and othersolids and filtration by 0.2 μm. It was transferred to a clear GC vialand 2-Ethylbutyric acid (Sigma) was used as the internal standard. Theconcentration of SCFA was analyzed using a Varian 3500 GCflame-ionization system, fitted with a with a ZB-FFAP column (30 m×0.32mm×0.25 mm; Phenomenex). A standard curve was built with differentconcentrations of a standard mix containing acetate, propionate,iso-butyrate, n-butyrate, isovalerate and valerate (Sigma). Peaks wereintegrated by using the Varian Star Chromatography Workstation version6.0 software. All SCFA data are expressed as μmol/g.

Statistical Analysis

Normally distributed data are presented as mean±SEM; Non-parametricdatasets are presented as median with inter-quartile range. Unpairedtwo-tailed t-test were applied to analyse parametric data andMann-Whitney test was used for non-parametric. Spearman's rankcorrelation coefficient was employed for the correlation analysis in thepooled datasets. A p value <0.05 was deemed significant in all cases.

Results—Neurotransmitter Production

The results in FIG. 23 show the effect of MRx005 treatment on theconcentration of neurotransmitters in the brain of mice. Most notably,treatment with MRx005 leads to a decrease in dopamine.

Results—Gene Expression

Expression of genes for neurotransmitter receptors [serotonin receptor1a(5-HTR1a), dopamine D1 receptor, GABA receptor subunit B1, GABAAreceptor, NMDA2A (Grin2A) and NMDA2B (Grin2b) receptor], inflammatorymarkers [IL-1β, IL6, CD11b, TNFα and TLR4], and endocrine markers[corticosterone releasing factor (CRF), corticosterone releasing factorreceptors 1 and 2 (CRFR1, CRFR2), brain-derived neurotrophin factor(BDNF), vasopressin receptor, oxytocin receptor, glucocorticoid receptorand mineralocorticoid receptor] were analysed in brain tissue from thehippocampus, amygdala and prefrontal cortex.

FIGS. 24-38 show the changes in gene expression after MRX005 or MRX0029treatment in the hippocampal, amygdala and prefrontal cortex. Treatmentwith MRx0029 led to an increase in glucocorticoid receptor expression inthe amygdala (FIG. 31C). FIG. 32A shows that MRx005 significantlyincreased the expression of BDNF in the amygdala, while treatment withMRx0029 significantly increased the expression of TLR4 in the amygdala(FIG. 32).

Both MRx005 and MRx0029 can increase expression of CD11b in the amygdala(FIG. 33A), while the expression of IL-6, Grin2a and Grin2b is reducedafter MRx005 treatment (FIGS. 33B-D). In addition, MRx005 and MRx0029significantly increased the expression of GABRA2 and increased theexpression of GABBR1 in the amygdala.

Treatment with MRx005 led to a significant increase in the expression ofBDNF in the prefrontal cortex (FIG. 35B).

Discussion

MRx005 and MRx0029 administration caused changes in gene expression,especially in the amygdala.

Results—Effect on Tph1 and IDO-1 expression

FIG. 39 shows that MRx0029 can significantly increase the expressiontryptophan hydroxylase-1 (Tph1) in the colon and that MRX005 treatmentcan increase IDO-1 expression in the colon. Treatment with MRX005increased the expression of Tph1 and IDO1 in the ileum (FIG. 40).

Indoleamine-pyrrole 2,3-dioxygenase-1 (IDO-1) the first andrate-limiting enzyme in the tryptophan/kynurenine pathway whiletryptophan hydroxylase 1 (Tph1), an isoform of the enzyme tryptophanhydroxylase, responsible for the synthesis of serotonin. These datasuggest that MRx0029 and MRx005 may affect serotonin levels and thetryptophan/kynurenine pathway.

Results—Effect on Tryptophan Metabolite Levels

FIG. 41 shows the effect of treatment with MRx005 on the levels ofcirculating kynurenine and tryptophan.

Results—Effect on Cytokine Expression from Splenocytes

The ex-vivo splenocyte assay involves challenging the splenocytes (cellsisolated from the spleen—a main organ involved in immune defence), witha bacterio- or viral-mimetic challenge.

MRX005 significantly reduced the levels of interferon-γ in splenocytesfollowing a challenge with LPS (FIG. 42). In addition, MRX005 reducedthe levels of interleukin-6 and tumour necrosis factor after a challengewith LPS (FIGS. 44 and 45, respectively). Treatment with MRx0029 led toa reduction in interferon-γ, interleukin-1β and interleukin-6 followinga challenge with LPS (FIGS. 42, 43 and 44, respectively).

Treatment with MRx005 and MRx0029 led to an increase in the levels ofthe chemoattractant CXCL1 (FIG. 47).

Results—Effect on Caecal Short Chain Fatty Acid Levels

Short chain fatty acids (SCFAs) are produced when non-digestible fibresfrom the diet are fermented by bacteria in the gut. The effects ofMRX005 administration are shown in FIG. 48.

Example 18—Further Analysis of MRX029 and MRX005-Induced Changes in GeneExpression Levels

Methods

Cell Line

SH-SY5Y cells

Bacterial Strains

-   -   755: Parabacteroides distasonis (MRX005)    -   Megasphaera massiliensis (MRX0029)

qPCR

SHSY5Y were plated in 10 cm petri dishes a density of 2×10⁶ cells. After24h cells were treated in differentiation medium (growth mediumcontaining 1% FBS without RA) with 10% bacteria supernatants or YCFA+,10 uM RA, 200 uM hexanoic acid or 200 uM valproic acid, for 17 hrs.There after representative images were taken using phase contrast EVOSXL core microscope at 40×/0.65 magnification. Cells were collected, andtotal RNA was isolated according to RNeasy mini kit protocol (Qiagen).cDNAs were made using the high capacity cDNA reverse transcription kit(Applied Biosystems). Gene expression was measured using qPCR. GAPDH wasused as internal control. Fold change was calculated according to the2^((−ΔΔct)) method. The primer sequences for MAP2, DRD2, GABRB3, SYP,PINK1, PARK? and NSE are provided in the sequence listing.

Immuno-Labelling and Cell Imaging

Cells were seeded onto 8-well chamber slides (Marienfeld LaboratoryGlassware) at 5×10⁴ cells/well overnight and were treated with 10%bacterial supernatant for 24 h. For differentiation, cells were treatedwith 10 nM RA for 5 days before treating with cell-free bacterialsupernatant for 24 h. Afterwards, the cells were fixed with 4%paraformaldehyde in PBS for 20 minutes at room temperature (RT). Fixedcells were washed with PBS, and permeabilized with 1% Triton X-100 inPBS for 10 minutes. After washing with PBS, the slides were incubatedwith blocking buffer (4% BSA/PBS) for 1 h at RT before adding anti-MAP2antibody or β3-tubulin (sc-74421 and sc-80005 respectively, Santa CruzBiotechnology Inc) diluted in 1% BSA/PBS for 12 h at 4° C. They werethen washed twice with PBS, followed by incubation with Alexa Flour 488conjugated anti-mouse (Molecular Probes Inc) and Alexa Flour 594conjugated Phalloidin (ab176757, Abcam) for 1 h at RT. After washing 3×with PBS, the slides were staining with DAPI and mounted withVectashield® (Vector Laboratories). Slides were viewed using a Axioskop50 microscope (Zeiss) equipped with a 63×/1.2 W Korr objective andfilter sets suitable for detection of the fluorochromes used. Manualexposure times for the digital acquisition of images immuno-labelledwith MAP-2 were kept constant allowing comparison between differentwells and treatments. Phalloidin (F-actin) and DAPI exposure timesvaried to suit the field of view. Randomised fields of view wereacquired using a Qlmaging camera controlled by Image Pro Plus software.Images were saved as TIFF files and opened in Adobe Photoshop CC2015.1.2. Images of the MAP-2, DAPI and Phalloidin images were thenoverlaid and merged. Representative images were selected to illustratethe differences in abundance and location of the proteins examined.

Immunoblotting

SH-SY5Y cells cultured under the indicated conditions described above,treated with MRx0005 and MRx0029 for 24h and then lysed in RIPA buffercontaining cocktail of protease inhibitors (Roche Diagnostics, UK).Protein concentration was estimated using the BCA protein assay kit(Pierce Biotechnology, Rockford, Ill.), separated by SDS-PAGE andtransferred to a PVDF membrane. Membranes were then blocked with 5%non-fat dry milk or 5% BSA and incubated overnight at 4° C. with theprimary antibodies (respectively MAP2 and β3-tubulin). The blots werethen incubated with the appropriate horseradish peroxidase(HRP)-conjugated secondary antibody, and proteins were detected bychemiluminescence detection kit (Pierce Biotechnology, Rockford, Ill.).For both MAP2 and β3-tubulin, β-actin served as a control to monitorprotein loading variability amongst samples.

Results and Discussion

Gene Expression

FIGS. 13a (graph insert) and 49 show the MRx0029 and MRX005-inducedchanges in expression levels of Actin, Villin, Occludin TJP1, TJP2,MAP2, DRD2, GABRB3, SYP, PINK′, PARK? and NSE.

Microscopy and Immunoblotting

FIG. 50 shows the change in the level of expression of MAP2 in SHSY5Ycells as determined by confocal microscopy. The expression levels ofMAP2 and B3-tubulin were also quantified by immunoblot analysis. Theresults shown in FIGS. 50M and N indicate that MRX029 induces anincrease in the level expression of MAP2

Sequences

SEQ ID NO:1 (Megasphaera massiliensis gene for 16S ribosomal RNA,partial sequence, strain: NP3-JX424772.1)

   1 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ttaacacatg caagtcgaac  61 gagaagagat gagaagcttg cttcttatca attcgagtgg caaacgggtg agtaacgcgt 121 aagcaacctg cccttcagat ggggacaaca gctggaaacg gctgctaata ccgaatacgt 181 tctttccgcc gcatgacggg aagaagaaag ggaggccttc gggctttcgc tggaggaggg 241 gcttgcgtct gattagctag ttggaggggt aacggcccac caaggcgacg atcagtagcc 301 ggtctgagag gatgaacggc cacattggga ctgagacacg gcccagactc ctacgggagg 361 cagcagtggg gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgaacga 421 tgacggcctt cgggttgtaa agttctgtta tatgggacga acagggcatc ggttaatacc 481 cggtgtcttt gacggtaccg taagagaaag ccacggctaa ctacgtgcca gcagccgcgg 541 taatacgtag gtggcaagcg ttgtccggaa ttattgggcg taaagggcgc gcaggcggca 601 tcgcaagtcg gtcttaaaag tgcggggctt aaccccgtga ggggaccgaa actgtgaagc 661 tcgagtgtcg gagaggaaag cggaattcct agtgtagcgg tgaaatgcgt agatattagg 721 aggaacacca gtggcgaaag cggctttctg gacgacaact gacgctgagg cgcgaaagcc 781 aggggagcaa acgggattag ataccccggt agtcctggcc gtaaacgatg gatactaggt 841 gtaggaggta tcgactcctt ctgtgccgga gttaacgcaa taagtatccc gcctggggag 901 tacggccgca aggctgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagtat 961 gtggtttaat tcgacgcaac gcgaagaacc ttaccaagcc ttgacattga ttgctacgga1021 aagagatttc cggttcttct tcggaagaca agaaaacagg tggtgcacgg ctgtcgtcag1081 ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccctat cttctgttgc1141 cagcacctcg ggtggggact cagaagagac tgccgcagac aatgcggagg aaggcgggga1201 tgacgtcaag tcatcatgcc ccttatggct tgggctacac acgtactaca atggctctta1261 atagagggac gcgaaggagc gatccggagc aaaccccaaa aacagagtcc cagttcggat1321 tgcaggctgc aactcgcctg catgaagcag gaatcgctag taatcgcagg tcagcatact1381 gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacgaa agtcattcac1441 acccgaagcc ggtgaggcaa ccgcaaggaa ccagccgtcg aaggtggggg cgatgattgg1501 ggtgaagtcg taacaaggt

SEQ ID NO:2 (consensus 16S rRNA sequence for Megasphaera massiliensisstrain MRx0029)

TGAGAAGCTTGCTTCTTATCGATTCTAGTGGCAAACGGGTGAGTAACGCGTAAGCAACCTGCCCTTCAGATGGGGACAACAGCTGGAAACGGCTGCTAATACCGAATACGTTCTTTCCGCCGCATGACGGGAAGAAGAAAGGGAGGCCTTCGGGCTTTCGCTGGAGGAGGGGCTTGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAACGATGACGGCCTTCGGGTTGTAAAGTTCTGTTATATGGGACGAACAGGACATCGGTTAATACCCGGTGTCTTTGACGGTACCGTAAGAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCGCGCAGGCGGCATCGCAAGTCGGTCTTAAAAGTGCGGGGCTTAACCCCGTGAGGGGACCGAAACTGTGAAGCTCGAGTGTCGGAGAGGAAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAAGCGGCTTTCTGGACGACAACTGACGCTGAGGCGCGAAAGCCAGGGGAGCAAACGGGATTAGATACCCCGGTAGTCCTGGCCGTAAACGATGGATACTAGGTGTAGGAGGTATCGACTCCTTCTGTGCCGGAGTTAACGCAATAAGTATCCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGACGCAACGCGAAGAACCTTACCAAGCCTTGACATTGATTGCTACGGAAAGAGATTTCCGGTTCTTCTTCGGAAGACAAGAAAACAGGTGGTGCACGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTCTGTTGCCAGCACCTCGGGTGGGGACTCAGAAGAGACTGCCGCAGACAATGCGGAGGAAGGCGGGGATGACGTCAAGTCATCATGCCCCTTATGGCTTGGGCTACACACGTACTACAATGGCTCTTAATAGAGGGAAGCGAAGGAGCGATCCGGAGCAAACCCCAAAAACAGAGTCCCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCAGGAATCGCTAGTAATCGCAGGTCAGCATACTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAAAGTCATTCACACCCGAAGCCGGTGAGGCAACCGCAAG

Primers used for qPCR (with SEQ ID NO in brackets)

Name Forward sequence Reverse sequence ACTB GATCAAGATCATTGCTCCTCTTGTCAAGAAAGGGTGTAAC (3) (4) GAPDH GGTATCGTGGAAGGACTCATGATGCCAGTGAGCTTCCCGTTC (5) (6) MAP2 CTCAGCACCGCTAACAGAGGCATTGGCGCTTCTCTCCTC (7) (8) Occlud- AAGAGGAATTTTGACACTGGGCCATGTACTCTTCACTTTC in (9) (10) TJ1 AAGTCACACTGGTGAAATCCCTCTTGCTGCCAAACTATCT (11) (12) TJP2 CCCTCCCCTGGATCAGGATGCCATCAAACTCGTCCATCA (13) (14) Villin CATTACCTGCTCTACGTTTGAGATGGACATAAGATGAGGTG (15) (16)

SEQ ID NO:17 (consensus 16S rRNA sequence for Parabacteroides distasonisstrain MRX0005)

AMCCGGGTGGCGACCGGCGCACGGGTGAGTAACGCGTATGCAACTTGCCTATCAGAGGGGGATAACCCGGCGAAAGTCGGACTAATACCGCATGAAGCAGGGATCCCGCATGGGAATATTTGCTAAAGATTCATCGCTGATAGATAGGCATGCGTTCCATTAGGCAGTTGGCGGGGTAACGGCCCACCAAACCGACGATGGATAGGGGTTCTGAGAGGAAGGTCCCCCACATTGGTACTGAGACACGGACCAAACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGTGAGCCTGAACCAGCCAAGTCGCGTGAGGGATGAAGGTTCTATGGATCGTAAACCTCTTTTATAAGGGAATAAAGTGCGGGACGTGTCCCGTTTTGTATGTACCTTATGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATCCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGTGCGTAGGCGGCCTTTTAAGTCAGCGGTGAAAGTCTGTGGCTCAACCATAGAATTGCCGTTGAAACTGGGAGGCTTGAGTATGTTTGAGGCAGGCGGAATGCGTGGTGTAGCGGTGAAATGCATAGATATCACGCAGAACCCCGATTGCGAAGGCAGCCTGCCAAGCCATTACTGACGCTGATGCACGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCAGTAAACGATGATCACTAGCTGTTTGCGATACACTGTAAGCGGCACAGCGAAAGCGTTAAGTGATCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGTTTGAACGCATTCGGACMGAKGTGGAAACACATTTTCTAGCAATAGCCATTTGCGAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATAACGAGCGCAACCCTTGCCACTAGTTACTAACAGGTAAAGCTGAGGACTCTGGTGGGACTGCCAGCGTAAGCTGCGAGGAAGGCGGGGATGACGTCAAATCAGCACGGCCCTTACATCCGGGGCGACACACGTGTTACAATGGCGTGGACAAAGGGAAGCCACCTGGCGACAGGGAGCGAATCCCCAAACCACGTCTCAGTTCGGATCGGAGTCTGCAACCCGACTCCGTGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCATGGCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGCCATGGGAGCCGGGGGTACCTGAAGTCCGTAACCGCGAGGATCGGCCTAGGGTAAAACTGGTGACTGGGGCTAAGTCGTACGGGG

Primers and probes used for ex vivo qPCR (with SEQ ID NO in brackets)

Ex vivo:

Name Forward sequence Reverse sequence Probe ACTBGAT TAC TGC TCT GGC TCC   GAC TCA TCG TAC TCC TGC TTG/56-FAM/CTG GCC TCA /ZEN/CTG TAG (18) (19) TCC ACC TTC C/3IABkFQ/ (20)GAPDH AAT GGT GAA GGT CGG TGT  GTG GAG TCA TAC TGG AAC ATG  /56-FAM/TGC AAA TGG /ZEN/CAG G (21) TAG (22) CCC TGG TG/3IABkFQ/ (23)BDNF GCT GCC TTG ATG TTT ACT   GCA ACC GAA GTA TGA AAT AAC /56-FAM/ACC AGG TGA /ZEN/GAA GAG TTG AC (24) CA (25)TGA TGA CCA TCC/3IABkFQ/ (26) IL6 AGC CAG AGT CCT TCA GAG TCC TTA GCC ACT CCT TCT GT  /56-FAM/CCT ACC CCA /ZEN/ATT TCC A (27) (28)AAT GCT CTC CT/3IABkFQ/ (29)

Additional primers used in qPCR (with SEQ ID NO in brackets)

Gene  ID Forward sequence Reverse sequence NSE CCCTGTATCGTAAGAACGGTGCCACCATTGATCACGTTGA (30) (31) PINK1 CCCAAGCAACTAGCCCCTCGGCAGCACATCAGGGTAGTC (32) (33) PARK7 GTAGCCGTGATGTGGTCATTTCTGTGCGCCCAGATTACCT (34) (35) SYP CTCGGCTTTGTGAAGGTGCTGGCTTCATGGCATCAACTTCA (36) (37)

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1.-23. (canceled)
 24. A pharmaceutical composition comprising alyophilized bacteria strain of the genus Megasphaera, wherein thebacteria strain comprises a 16S rRNA gene sequence that has at least 96%sequence identity to the polynucleotide sequence of SEQ ID NO:2, asdetermined by a Smith-Waterman homology search algorithm using an affinegap search with a gap open penalty of 12, a gap extension penalty of 2,and a Blocks Substitution Matrix (BLOSUM) of 62, and a pharmaceuticallyacceptable excipient, diluent, or carrier.
 25. The pharmaceuticalcomposition of claim 24, wherein the therapeutically effective amount ofthe bacteria strain comprises from about 1×10³ to about 1×10¹¹ colonyforming units (CFU)/g of the bacteria strain with respect to totalweight of the pharmaceutical composition.
 26. The pharmaceuticalcomposition of claim 24, wherein the pharmaceutical compositioncomprises de minimis amounts of other bacteria strains.
 27. Thepharmaceutical composition of claim 24, wherein the pharmaceuticalcomposition is formulated for oral, rectal, nasal, buccal, sublingual,or subcutaneous administration.
 28. The pharmaceutical composition ofclaim 24, wherein the pharmaceutical composition is formulated fordelivery to an intestine of the subject.
 29. The pharmaceuticalcomposition of claim 24, wherein the pharmaceutical composition isencapsulated.
 30. The pharmaceutical composition of claim 24, whereinsaid pharmaceutical composition comprises an enteric coating.
 31. Thepharmaceutical composition of claim 24, further comprising an additionaltherapeutic agent.
 32. The pharmaceutical composition of claim 24,wherein the bacteria strain comprises a 16S rRNA gene sequence that hasat least 99% sequence identity to the polynucleotide sequence of SEQ IDNO:2, as determined by a Smith-Waterman homology search algorithm usingan affine gap search with a gap open penalty of 12, a gap extensionpenalty of 2, and a Blocks Substitution Matrix (BLOSUM) of
 62. 33. Thepharmaceutical composition of claim 24, wherein the bacteria straincomprises a 16S rRNA gene sequence that is the polynucleotide sequenceof SEQ ID NO:2.
 34. The pharmaceutical composition of claim 24, whereinthe bacteria strain is of the species Megasphaera massiliensis.
 35. Thepharmaceutical composition of claim 24, wherein the bacteria strain isthe bacteria strain deposited under accession number NCIMB
 42787. 36. Amethod of treating a neurological condition associated withneuroinflammation, oxidative stress, or neurodegeneration in a subjectin need thereof comprising: administering to the subject apharmaceutical composition comprising a therapeutically effective amountof a bacteria strain of the genus Megasphaera, wherein the bacteriastrain comprises a 16S rRNA gene sequence that has at least 95% sequenceidentity to the polynucleotide sequence of SEQ ID NO:2, as determined bya Smith-Waterman homology search algorithm using an affine gap searchwith a gap open penalty of 12, a gap extension penalty of 2, and aBlocks Substitution Matrix (BLOSUM) of 62, thereby treating theneurological condition associated with neuroinflammation, oxidativestress, or neurodegeneration in the subject.
 37. The method of claim 36,wherein the bacteria strain is of the species Megasphaera massiliensis.38. The method of claim 36, wherein the bacteria strain is the bacteriastrain deposited under accession number NCIMB
 42787. 39. The method ofclaim 36, wherein said administering comprises oral, rectal, nasal,buccal, sublingual, or subcutaneous administration.
 40. The method ofclaim 36, wherein the bacteria strain comprises a 16S rRNA gene sequencethat has at least 99% sequence identity to the polynucleotide sequenceof SEQ ID NO:2, as determined by a Smith-Waterman homology searchalgorithm using an affine gap search with a gap open penalty of 12, agap extension penalty of 2, and a Blocks Substitution Matrix (BLOSUM) of62.
 41. The method of claim 36, wherein the pharmaceutical compositionis encapsulated.
 42. The method of claim 36, wherein the bacteria strainis lyophilized.
 43. The method of claim 36, wherein the subject ishuman.